Compositions against cancer antigen liv-1 and uses thereof

ABSTRACT

Described herein are methods and compositions that can be used for diagnosis and treatment of cancer.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/528,602, filed Jun. 20, 2012, which is a continuation of U.S. patentapplication Ser. No. 12/622,298, filed Nov. 19, 2009, now abandoned,which is a continuation of U.S. patent application Ser. No. 11/962,593,filed Dec. 21, 2007, now abandoned, which is a continuation of U.S.patent application Ser. No. 10/769,612, filed Jan. 29, 2004, nowabandoned, which claims the benefit of U.S. Provisional Application No.60/443,712, filed Jan. 29, 2003, each application of which is herebyincorporated by reference in its entirety for all purposes.

REFERENCE TO A SEQUENCE LISTING

This application includes a sequence listing as a text file, named“0710-00115US_ST25.txt” created Aug. 26, 2009 and containing 25,269bytes. The material contained in this text file is incorporated byreference in its entirety for all purposes.

FIELD OF THE INVENTION

The invention relates to the identification and generation of antibodiesthat specifically bind to LIV-1 proteins; and to the use of suchantibodies and compositions comprising them, in the diagnosis,prognosis, and therapy of cancer.

BACKGROUND OF THE INVENTION

Zinc plays an essential role in cell growth, and is a cofactor of over300 enzymes, including enzymes important in angiogenesis and cellremodeling. Vallee, B. L., Auld, D. S., Biochem. 29:5647-5659 (1990).Zinc associates with many macromolecules in cells, including molecularcomponents that act to control growth, apoptosis, development anddifferentiation. Control of intracellular zinc levels, therefore, may beimportant in preventing the triggering of a variety of disease states,including cancer.

LIV-1 is a member of the LZT (LIV-1-ZIP Zinc Transporters) subfamily ofzinc transporter proteins. Taylor, K. M. and Nicholson, R. I., BiochimBiophys. Acta 1611:16-30 (2003). Computer analysis of the LIV-1 proteinreveals a potential metalloprotease motif, fitting the consensussequence for the catalytic zinc-binding site motif of the zincinmetalloprotease.

The structure of LIV-1 implicates a role for the protein as azinc-influx transporter protein. Experiments with recombinant LIV-1localizes the protein to the plasma membrane, similarly concentrated inlamellipodiae as membrane-type metalloproteases. Taylor and Nicholson,supra. Computer analysis predicts six to eight transmembrane domains, along extracellular N terminus, a short extracellular C terminus, as wellas the consensus sequence for the catalytic zinc-binding site ofmetalloproteases. LIV-1 distribution studies indicates primaryexpression in breast, prostate, pituitary gland and brain tissue. Taylorand Nicholson, supra.

The LIV-1 protein has also been implicated in certain cancerousconditions, e.g. breast cancer and prostate cancer. The detection ofLIV-1 is associated with estrogen receptor-positive breast cancer,McClelland, R. A., et al., Br. J. Cancer 77:1653-1656 (1998), and themetastatic spread of these cancers to the regional lymph nodes. Manning,D. A. et al., Eur. J. Cancer 30A:675-678 (1994). Antibodies useful fordiagnosis, prognosis, and effective treatment of cancer, includingmetastatic cancer, would be desirable. Accordingly, provided herein arecompositions and methods that can be used in diagnosis, prognosis, andtherapy of certain cancers.

SUMMARY OF THE INVENTION

The present invention provides anti-LIV-1 antibodies that are useful formaking conjugated antibodies for therapeutic purposes. For example, theanti-LIV-1 antibodies of the invention are useful as selective cytotoxicagents for LIV-1 expressing cells. In some embodiments, the antibodiesof the present invention are therapeutically useful in persons diagnosedwith cancer and other proliferative conditions, including benignproliferative conditions. In one aspect, the antibodies of the presentinvention can be used to treat proliferative conditions of the prostateor breast including, for example, prostate cancer or breast cancer.

The present invention provides antibodies that competitively inhibitbinding of proteins encoded by vectors containing some or all of thesequence associated with LIV-1 (Hs.79136). In some embodiments theantibodies are further conjugated to an effector component. The effectorcomponent can be a label (e.g., a fluorescent label, an effector domaine.g. MicA) or can be a cytotoxic moiety (e.g., a radioisotope or acytotoxic chemical). An exemplary cytotoxic chemical is auristatin-E. Inother embodiments the antibodies can be used alone to inhibit tumor cellgrowth.

The antibodies of the invention can be whole antibodies or can beantibody fragments. In some embodiments the immunoglobulin is ahumanized antibody. An exemplary antibody of the invention is defined byCDRs.

The invention further provides immunoassays using the immunoglobulins ofthe invention. These methods involve detecting a cancer cell in abiological sample from a patient by contacting the biological samplewith an antibody of the invention. The antibody is typically conjugatedto a label such as a fluorescent or other label.

The invention also provides double-stranded ribonucleic acids that bindto mRNA encoded by the LIV-1 nucleic acid of SEQ ID NO:1. Thedouble-stranded ribonucleic acids may cover the length of the targetmRNA, or may be short double-stranded ribonucleic acids complementary tothe target mRNA, e.g. siRNA.

The invention also provides pharmaceutical compositions comprising apharmaceutically acceptable excipient and the antibody or doublestranded ribonucleic acid of the invention. In these embodiments, theantibody can be further conjugated to an effector component. Theeffector component can be a label (e.g., a fluorescent label) or can bea cytotoxic moiety (e.g., a radioisotope or a cytotoxic chemical). Anexemplary cytotoxic chemical is auristatin-E. The antibodies in thepharmaceutical compositions can be whole antibodies or antibodyfragments. In some embodiments the immunoglobulin is a humanizedantibody.

The invention also provides methods of inhibiting proliferation of aprostate cancer-associated or breast cancer-associated cell. The methodcomprises contacting the cell with an antibody or double-strandedribonucleic acid of the invention. In most embodiments, the cancer cellis in a patient, typically a human. The patient may be undergoing atherapeutic regimen to treat metastatic or benign prostate cancer orbreast cancer or may be suspected of having prostate cancer or breastcancer.

DESCRIPTION OF THE TABLES AND FIGURES

Table 1 provides the cDNA (SEQ ID NO:1) and protein sequence for LIV-1(SEQ ID NO:2).

Table 2 provides DNA and peptide sequences for the LIV-1 antibody,#1.7A4 (SEQ ID NOS:3-6).

Table 3 provides a partial list of the variety of medical conditionsthat LIV-1 may be implicated in.

Table 4 provides a list of cell lines that may be used to validateanti-LIV-1 compositions in ovarian and bladder systems.

Table 5 provides LIV-1 mutant (BCR4MD cDNA (5A) and protein sequences(5B). Mutated residues are underlined.

Table 6 provides a list of antibodies generated against the LIV-1protein.

FIG. 1 shows a graph of the reduction in size of a prostate tumor invivo after Auristatin-E-conjugated LIV-1 antibody treatment.

FIG. 2 shows a graph of the reduction in size of a breast cancer tumorin vivo after Auristatin-E-conjugated LIV-1 antibody treatment.

FIG. 3 shows fluorescence micrograph images of LIV-1 antibody stainedtissue sections from breast cancer (left) and other normal tissues.

FIG. 4A shows a bar graph of the effect of a LIV-1 RNAi composition onMX-1 carcinoma cell growth in a clonogenic assay 14 days after additionof the LIV-1 siRNA.

FIG. 4B shows a bar graph of the effect of a LIV-1 RNAi composition hason MX-1 carcinoma cell growth in a clonogenic assay 17 days afteraddition of the LIV-1 siRNA.

FIG. 5 shows a fluorescence microscope image of HCT116 cells transfectedwithout (FIG. 5A) or with (FIG. 5B) a LIV-1 siRNA.

FIG. 6 shows a bar graph of the binding strength of various LIV-1antibodies on LIV-1 expressing cells (MX-1 breast carcinoma cells).

FIG. 7 shows the inhibition of several LIV-1 antibodies on epithelialovarian carcinoma cell growth (CSOC), as compared to an isotype IgG1control.

FIG. 8 shows the inhibition of several LIV-1 antibodies on mammarycarcinoma cell growth (MX-1), as compared to an isotype IgG1 control.

FIG. 9 shows the inhibition of several LIV-1 antibodies on prostatecarcinoma cell growth (LNCaP), as compared to an isotype IgG1 control.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel reagents and methods for treatment,diagnosis and prognosis for certain cancers using antibodies anddouble-stranded ribonucleic acids against LIV-1. In particular, thepresent invention provides anti-LIV-1 antibodies that are particularlyuseful as selective cytotoxic agents for LIV-1 expressing cells.

Epitope mapping of antibodies showing high affinity binding can becarried out through competitive binding analyses. Using thismethodology, antibodies recognizing a number of individual epitopes canbe identified. The antibodies are then assessed for LIV-1 dependent celldeath in vitro. Using these methods antibodies that promote cell deathcan be identified.

DEFINITIONS

As used herein, “antibody” includes reference to an immunoglobulinmolecule immunologically reactive with a particular antigen, andincludes both polyclonal and monoclonal antibodies. The term alsoincludes genetically engineered forms such as chimeric antibodies (e.g.,humanized murine antibodies) and heteroconjugate antibodies (e.g.,bispecific antibodies). The term “antibody” also includes antigenbinding forms of antibodies, including fragments with antigen-bindingcapability (e.g., Fab′, F(ab′)₂, Fab, Fv and rIgG. See also, PierceCatalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.).See also, e.g., Kuby, J., Immunology, 3^(rd) Ed., W.H. Freeman & Co.,New York (1998). The term also refers to recombinant single chain Fvfragments (scFv). The term antibody also includes bivalent or bispecificmolecules, diabodies, triabodies, and tetrabodies. Bivalent andbispecific molecules are described in, e.g., Kostelny et al. (1992) JImmunol 148:1547, Pack and Pluckthun (1992) Biochemistry 31:1579,Hollinger et al., 1993, supra, Gruber et al. (1994) J. Immunol.:5368,Zhu et al. (1997) Protein Sci 6:781, Hu et al. (1996) Cancer Res.56:3055, Adams et al. (1993) Cancer Res. 53:4026, and McCartney, et al.(1995) Protein Eng. 8:301.

An antibody immunologically reactive with a particular antigen can begenerated by recombinant methods such as selection of libraries ofrecombinant antibodies in phage or similar vectors, see, e.g., Huse etal., Science 246:1275-1281 (1989); Ward et al., Nature 341:544-546(1989); and Vaughan et al., Nature Biotech. 14:309-314 (1996), or byimmunizing an animal with the antigen or with DNA encoding the antigen.

Typically, an immunoglobulin has a heavy and light chain. Each heavy andlight chain contains a constant region and a variable region, (theregions are also known as “domains”). Light and heavy chain variableregions contain four “framework” regions interrupted by threehypervariable regions, also called “complementarity-determining regions”or “CDRs”. The extent of the framework regions and CDRs have beendefined. The sequences of the framework regions of different light orheavy chains are relatively conserved within a species. The frameworkregion of an antibody, that is the combined framework regions of theconstituent light and heavy chains, serves to position and align theCDRs in three dimensional space.

The CDRs are primarily responsible for binding to an epitope of anantigen. The CDRs of each chain are typically referred to as CDR1, CDR2,and CDR3, numbered sequentially starting from the N-terminus, and arealso typically identified by the chain in which the particular CDR islocated. Thus, a V_(H) CDR3 is located in the variable domain of theheavy chain of the antibody in which it is found, whereas a V_(L) CDR1is the CDR1 from the variable domain of the light chain of the antibodyin which it is found.

References to “V_(H)” or a “VH” refer to the variable region of animmunoglobulin heavy chain of an antibody, including the heavy chain ofan Fv, scFv, or Fab. References to “V_(L)” or a “VL” refer to thevariable region of an immunoglobulin light chain, including the lightchain of an Fv, scFv, dsFv or Fab.

The phrase “single chain Fv” or “scFv” refers to an antibody in whichthe variable domains of the heavy chain and of the light chain of atraditional two chain antibody have been joined to form one chain.Typically, a linker peptide is inserted between the two chains to allowfor proper folding and creation of an active binding site.

A “chimeric antibody” is an immunoglobulin molecule in which (a) theconstant region, or a portion thereof, is altered, replaced or exchangedso that the antigen binding site (variable region) is linked to aconstant region of a different or altered class, effector functionand/or species, or an entirely different molecule which confers newproperties to the chimeric antibody, e.g., an enzyme, toxin, hormone,growth factor, drug, etc.; or (b) the variable region, or a portionthereof, is altered, replaced or exchanged with a variable region havinga different or altered antigen specificity.

A “humanized antibody” is an immunoglobulin molecule which containsminimal sequence derived from non-human immunoglobulin. Humanizedantibodies include human immunoglobulins (recipient antibody) in whichresidues from a complementary determining region (CDR) of the recipientare replaced by residues from a CDR of a non-human species (donorantibody) such as mouse, rat or rabbit having the desired specificity,affinity and capacity. In some instances, Fv framework residues of thehuman immunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, a humanized antibody will comprise substantiallyall of at least one, and typically two, variable domains, in which allor substantially all of the CDR regions correspond to those of anon-human immunoglobulin and all or substantially all of the framework(FR) regions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin (Jones et al., Nature 321:522-525 (1986); Riechmann etal., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992)). Humanization can be essentially performed followingthe method of Winter and co-workers (Jones et al., Nature 321:522-525(1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al.,Science 239:1534-1536 (1988)), by substituting rodent CDRs or CDRsequences for the corresponding sequences of a human antibody.Accordingly, such humanized antibodies are chimeric antibodies (U.S.Pat. No. 4,816,567), wherein substantially less than an intact humanvariable domain has been substituted by the corresponding sequence froma non-human species.

“Epitope” or “antigenic determinant” refers to a site on an antigen towhich an antibody binds. Epitopes can be formed both from contiguousamino acids or noncontiguous amino acids juxtaposed by tertiary foldingof a protein. Epitopes formed from contiguous amino acids are typicallyretained on exposure to denaturing solvents whereas epitopes formed bytertiary folding are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, and more usually, atleast 5 or 8-10 amino acids in a unique spatial conformation. Methods ofdetermining spatial conformation of epitopes include, for example, x-raycrystallography and 2-dimensional nuclear magnetic resonance. See, e.g.,Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66,Glenn E. Morris, Ed (1996).

The term “LIV-1 protein” or “LIV-1 polynucleotide” refers to nucleicacid and polypeptide polymorphic variants, alleles, mutants, andinterspecies homologues that: (1) have a nucleotide sequence that hasgreater than about 60% nucleotide sequence identity, 65%, 70%, 75%, 80%,85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% orgreater nucleotide sequence identity, preferably over a region of atleast about 25, 50, 100, 200, 500, 1000, or more nucleotides, to anucleotide sequence of SEQ ID NO:1; (2) bind to antibodies, e.g.,polyclonal antibodies, raised against an immunogen comprising an aminoacid sequence encoded by a nucleotide sequence of SEQ ID NO:1, andconservatively modified variants thereof; (3) specifically hybridizeunder stringent hybridization conditions to a nucleic acid sequence, orthe complement thereof of SEQ ID NO:1 and conservatively modifiedvariants thereof or (4) have an amino acid sequence that has greaterthan about 60% amino acid sequence identity, 65%, 70%, 75%, 80%, 85%,90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greateramino sequence identity, preferably over a region of at least about 25,50, 100, 200, or more amino acids, to an amino acid sequence of SEQ IDNO:2. A polynucleotide or polypeptide sequence is typically from amammal including, but not limited to, primate, e.g., human; rodent,e.g., rat, mouse, hamster; cow, pig, horse, sheep, or other mammal. A“LIV-1 polypeptide” and a “LIV-1 polynucleotide,” include both naturallyoccurring or recombinant forms.

A “full length” LIV-1 protein or nucleic acid refers to a prostatecancer or breast cancer polypeptide or polynucleotide sequence, or avariant thereof, that contains all of the elements normally contained inone or more naturally occurring, wild type LIV-1 polynucleotide orpolypeptide sequences. For example, a full length LIV-1 nucleic acidwill typically comprise all of the exons that encode for the fulllength, naturally occurring protein. The “full length” may be prior to,or after, various stages of post-translation processing or splicing,including alternative splicing.

“Biological sample” as used herein is a sample of biological tissue orfluid that contains nucleic acids or polypeptides, e.g., of a LIV-1protein, polynucleotide or transcript. Such samples include, but are notlimited to, tissue isolated from primates, e.g., humans, or rodents,e.g., mice, and rats. Biological samples may also include sections oftissues such as biopsy and autopsy samples, frozen sections taken forhistologic purposes, blood, plasma, serum, sputum, stool, tears, mucus,hair, skin, etc. Biological samples also include explants and primaryand/or transformed cell cultures derived from patient tissues. Abiological sample is typically obtained from a eukaryotic organism, mostpreferably a mammal such as a primate e.g., chimpanzee or human; cow;dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird;reptile; or fish.

“Providing a biological sample” means to obtain a biological sample foruse in methods described in this invention. Most often, this will bedone by removing a sample of cells from an animal, but can also beaccomplished by using previously isolated cells (e.g., isolated byanother person, at another time, and/or for another purpose), or byperforming the methods of the invention in vivo. Archival tissues,having treatment or outcome history, will be particularly useful.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 60% identity, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specifiedregion, when compared and aligned for maximum correspondence over acomparison window or designated region) as measured using a BLAST orBLAST 2.0 sequence comparison algorithms with default parametersdescribed below, or by manual alignment and visual inspection (see,e.g., NCBI web site or the like). Such sequences are then said to be“substantially identical.” This definition also refers to, or may beapplied to, the compliment of a test sequence. The definition alsoincludes sequences that have deletions and/or additions, as well asthose that have substitutions, as well as naturally occurring, e.g.,polymorphic or allelic variants, and man-made variants. As describedbelow, algorithms can account for gaps and the like. Preferably,identity exists over a region that is at least about 25 amino acids ornucleotides in length, or more preferably over a region that is 50-100amino acids or nucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof one of the number of contiguous positions selected from the groupconsisting typically of from 20 to 600, usually about 50 to about 200,more usually about 100 to about 150 in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned. Methods of alignment ofsequences for comparison are well-known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by thehomology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443(1970), by the search for similarity method of Pearson & Lipman, Proc.Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Dr.,Madison, Wis.), or by manual alignment and visual inspection (see, e.g.,Current Protocols in Molecular Biology (Ausubel et al., eds. 1995supplement)).

Examples of algorithms that are suitable for determining percentsequence identity and sequence similarity include the BLAST and BLAST2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res.25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990). BLAST and BLAST 2.0 are used, with the parameters describedherein, to determine percent sequence identity for the nucleic acids andproteins of the invention. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation. This algorithm involves first identifying high scoringsequence pairs (HSPs) by identifying short words of length W in thequery sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al., supra). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are extended in both directions along each sequencefor as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, e.g., for nucleotide sequences,the parameters M (reward score for a pair of matching residues;always >0) and N (penalty score for mismatching residues; always <0).For amino acid sequences, a scoring matrix is used to calculate thecumulative score. Extension of the word hits in each direction arehalted when: the cumulative alignment score falls off by the quantity Xfrom its maximum achieved value; the cumulative score goes to zero orbelow, due to the accumulation of one or more negative-scoring residuealignments; or the end of either sequence is reached. The BLASTalgorithm parameters W, T, and X determine the sensitivity and speed ofthe alignment. The BLASTN program (for nucleotide sequences) uses asdefaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=−4 anda comparison of both strands. For amino acid sequences, the BLASTPprogram uses as defaults a wordlength of 3, and expectation (E) of 10,and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl.Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of10, M=5, N=−4, and a comparison of both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001. Log valuesmay be large negative numbers, e.g., 5, 10, 20, 30, 40, 40, 70, 90, 110,150, 170, etc.

An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, e.g., where the two peptides differonly by conservative substitutions. Another indication that two nucleicacid sequences are substantially identical is that the two molecules ortheir complements hybridize to each other under stringent conditions, asdescribed below. Yet another indication that two nucleic acid sequencesare substantially identical is that the same primers can be used toamplify the sequences.

A “host cell” is a naturally occurring cell or a transformed cell thatcontains an expression vector and supports the replication or expressionof the expression vector. Host cells may be cultured cells, explants,cells in vivo, and the like. Host cells may be prokaryotic cells such asE. coli, or eukaryotic cells such as yeast, insect, amphibian, ormammalian cells such as CHO, HeLa, and the like (see, e.g., the AmericanType Culture Collection catalog).

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is substantially or essentially free from components thatnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. A protein or nucleic acid that is thepredominant species present in a preparation is substantially purified.In particular, an isolated nucleic acid is separated from some openreading frames that naturally flank the gene and encode proteins otherthan protein encoded by the gene. The term “purified” in someembodiments denotes that a nucleic acid or protein gives rise toessentially one band in an electrophoretic gel. Preferably, it meansthat the nucleic acid or protein is at least 85% pure, more preferablyat least 95% pure, and most preferably at least 99% pure. “Purify” or“purification” in other embodiments means removing at least onecontaminant from the composition to be purified. In this sense,purification does not require that the purified compound be homogenous,e.g., 100% pure.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers, those containing modified residues, and non-naturallyoccurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction similarly to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers tocompounds that have the same basic chemical structure as a naturallyoccurring amino acid, e.g., an α carbon that is bound to a hydrogen, acarboxyl group, an amino group, and an R group, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. Suchanalogs may have modified R groups (e.g., norleucine) or modifiedpeptide backbones, but retain the same basic chemical structure as anaturally occurring amino acid. Amino acid mimetics refers to chemicalcompounds that have a structure that is different from the generalchemical structure of an amino acid, but that functions similarly to anaturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical or associated, e.g., naturallycontiguous, sequences. Because of the degeneracy of the genetic code, alarge number of functionally identical nucleic acids encode mostproteins. For instance, the codons GCA, GCC, GCG, and GCU all encode theamino acid alanine. Thus, at every position where an alanine isspecified by a codon, the codon can be altered to another of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations,” which are onespecies of conservatively modified variations. Every nucleic acidsequence herein which encodes a polypeptide also describes silentvariations of the nucleic acid. One of skill will recognize that incertain contexts each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, often silent variations of a nucleicacid which encodes a polypeptide is implicit in a described sequencewith respect to the expression product, but not with respect to actualprobe sequences.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention. Typically conservativesubstitutions for one another: 1) Alanine (A), Glycine (G); 2) Asparticacid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4)Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine(M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7)Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see,e.g., Creighton, Proteins (1984)).

Macromolecular structures such as polypeptide structures can bedescribed in terms of various levels of organization. For a generaldiscussion of this organization, see, e.g., Alberts et al., MolecularBiology of the Cell (3rd ed., 1994) and Cantor & Schimmel, BiophysicalChemistry Part I: The Conformation of Biological Macromolecules (1980).“Primary structure” refers to the amino acid sequence of a particularpeptide. “Secondary structure” refers to locally ordered, threedimensional structures within a polypeptide. These structures arecommonly known as domains. Domains are portions of a polypeptide thatoften form a compact unit of the polypeptide and are typically 25 toapproximately 500 amino acids long. Typical domains are made up ofsections of lesser organization such as stretches of (-sheet and(-helices. “Tertiary structure” refers to the complete three dimensionalstructure of a polypeptide monomer. “Quaternary structure” refers to thethree dimensional structure formed, usually by the noncovalentassociation of independent tertiary units. Anisotropic terms are alsoknown as energy terms.

A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, chemical, orother physical means. For example, useful labels include fluorescentdyes, electron-dense reagents, enzymes (e.g., as commonly used in anELISA), biotin, digoxigenin, or haptens and proteins or other entitieswhich can be made detectable, e.g., by incorporating a radiolabel intothe peptide or used to detect antibodies specifically reactive with thepeptide. The radioisotope may be, for example, ³H, ¹⁴C, ³²P, ³⁵S, or¹²⁵I. In some cases, particularly using antibodies against the proteinsof the invention, the radioisotopes are used as toxic moieties, asdescribed below. The labels may be incorporated into the LIV-1 nucleicacids, proteins and antibodies at any position. Any method known in theart for conjugating the antibody to the label may be employed, includingthose methods described by Hunter et al., Nature, 144:945 (1962); Davidet al., Biochemistry, 13:1014 (1974); Pain et al., J. Immunol. Meth.,40:219 (1981); and Nygren, J. Histochem. and Cytochem., 30:407 (1982).The lifetime of radiolabeled peptides or radiolabeled antibodycompositions may extended by the addition of substances that stablizethe radiolabeled peptide or antibody and protect it from degradation.Any substance or combination of substances that stablize theradiolabeled peptide or antibody may be used including those substancesdisclosed in U.S. Pat. No. 5,961,955.

An “effector” or “effector moiety” or “effector component” is a moleculethat is bound (or linked, or conjugated), either covalently, through alinker or a chemical bond, or noncovalently, through ionic, van derWaals, electrostatic, or hydrogen bonds, to an antibody. The “effector”can be a variety of molecules including, e.g., detection moietiesincluding radioactive compounds, fluorescent compounds, an enzyme orsubstrate, tags such as epitope tags, a toxin; activatable moieties, achemotherapeutic agent; a chemoattractant, a lipase; an antibiotic; or aradioisotope emitting “hard” e.g., beta radiation.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, e.g., recombinant cells express genes that are not foundwithin the native (non-recombinant) form of the cell or express nativegenes that are otherwise abnormally expressed, under expressed or notexpressed at all. By the term “recombinant nucleic acid” herein is meantnucleic acid, originally formed in vitro, in general, by themanipulation of nucleic acid, e.g., using polymerases and endonucleases,in a form not normally found in nature. In this manner, operably linkageof different sequences is achieved. Thus an isolated nucleic acid, in alinear form, or an expression vector formed in vitro by ligating DNAmolecules that are not normally joined, are both considered recombinantfor the purposes of this invention. It is understood that once arecombinant nucleic acid is made and reintroduced into a host cell ororganism, it will replicate non-recombinantly, i.e., using the in vivocellular machinery of the host cell rather than in vitro manipulations;however, such nucleic acids, once produced recombinantly, althoughsubsequently replicated non-recombinantly, are still consideredrecombinant for the purposes of the invention. Similarly, a “recombinantprotein” is a protein made using recombinant techniques, e.g., throughthe expression of a recombinant nucleic acid as depicted above.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not normally found in the same relationship toeach other in nature. For instance, the nucleic acid is typicallyrecombinantly produced, having two or more sequences, e.g., fromunrelated genes arranged to make a new functional nucleic acid, e.g., apromoter from one source and a coding region from another source.Similarly, a heterologous protein will often refer to two or moresubsequences that are not found in the same relationship to each otherin nature (e.g., a fusion protein).

A “promoter” is defined as an array of nucleic acid control sequencesthat direct transcription of a nucleic acid. As used herein, a promoterincludes necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A “constitutive”promoter is a promoter that is active under most environmental anddevelopmental conditions. An “inducible” promoter is a promoter that isactive under environmental or developmental regulation. The term“operably linked” refers to a functional linkage between a nucleic acidexpression control sequence (such as a promoter, or array oftranscription factor binding sites) and a second nucleic acid sequence,wherein the expression control sequence directs transcription of thenucleic acid corresponding to the second sequence.

An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein, in a heterogeneous population ofproteins and other biologics. Thus, under designated immunoassayconditions, the specified antibodies bind to a particular proteinsequences at least two times the background and more typically more than10 to 100 times background.

Specific binding to an antibody under such conditions requires anantibody that is selected for its specificity for a particular protein.For example, polyclonal antibodies raised to a particular protein,polymorphic variants, alleles, orthologs, and conservatively modifiedvariants, or splice variants, or portions thereof, can be selected toobtain only those polyclonal antibodies that are specificallyimmunoreactive with LIV-1 and not with other proteins. This selectionmay be achieved by subtracting out antibodies that cross-react withother molecules. A variety of immunoassay formats may be used to selectantibodies specifically immunoreactive with a particular protein. Forexample, solid-phase ELISA immunoassays are routinely used to selectantibodies specifically immunoreactive with a protein (see, e.g., Harlow& Lane, Antibodies, A Laboratory Manual (1988) for a description ofimmunoassay formats and conditions that can be used to determinespecific immunoreactivity).

“Tumor cell” refers to precancerous, cancerous, and normal cells in atumor.

“Cancer cells,” “transformed” cells or “transformation” in tissueculture, refers to spontaneous or induced phenotypic changes that do notnecessarily involve the uptake of new genetic material. Althoughtransformation can arise from infection with a transforming virus andincorporation of new genomic DNA, or uptake of exogenous DNA, it canalso arise spontaneously or following exposure to a carcinogen, therebymutating an endogenous gene. Transformation is associated withphenotypic changes, such as immortalization of cells, aberrant growthcontrol, nonmorphological changes, and/or malignancy (see, Freshney,Culture of Animal Cells a Manual of Basic Technique (3rd ed. 1994)).

Expression of LIV-1 Polypeptides from Nucleic Acids

Nucleic acids of the invention can be used to make a variety ofexpression vectors to express LIV-1 polypeptides which can then be usedto raise antibodies of the invention, as described below. Expressionvectors and recombinant DNA technology are well known to those of skillin the art and are used to express proteins. The expression vectors maybe either self-replicating extrachromosomal vectors or vectors whichintegrate into a host genome. Generally, these expression vectorsinclude transcriptional and translational regulatory nucleic acidoperably linked to the nucleic acid encoding the LIV-1 protein. The term“control sequences” refers to DNA sequences used for the expression ofan operably linked coding sequence in a particular host organism.Control sequences that are suitable for prokaryotes, e.g., include apromoter, optionally an operator sequence, and a ribosome binding site.Eukaryotic cells are known to utilize promoters, polyadenylationsignals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is typicallyaccomplished by ligation at convenient restriction sites. If such sitesdo not exist, synthetic oligonucleotide adaptors or linkers are used inaccordance with conventional practice. Transcriptional and translationalregulatory nucleic acid will generally be appropriate to the host cellused to express the LIV-1 protein. Numerous types of appropriateexpression vectors, and suitable regulatory sequences are known in theart for a variety of host cells.

In general, transcriptional and translational regulatory sequences mayinclude, but are not limited to, promoter sequences, ribosomal bindingsites, transcriptional start and stop sequences, translational start andstop sequences, and enhancer or activator sequences. In a oneembodiment, the regulatory sequences include a promoter andtranscriptional start and stop sequences.

Promoter sequences encode either constitutive or inducible promoters.The promoters may be either naturally occurring promoters or hybridpromoters. Hybrid promoters, which combine elements of more than onepromoter, are also known in the art, and are useful in the presentinvention.

In addition, an expression vector may comprise additional elements. Forexample, the expression vector may have two replication systems, thusallowing it to be maintained in two organisms, e.g. in mammalian orinsect cells for expression and in a prokaryotic host for cloning andamplification. Furthermore, for integrating expression vectors, theexpression vector contains at least one sequence homologous to the hostcell genome, and preferably two homologous sequences which flank theexpression construct. The integrating vector may be directed to aspecific locus in the host cell by selecting the appropriate homologoussequence for inclusion in the vector. Constructs for integrating vectorsare well known in the art (e.g., Fernandez & Hoeffler, supra).

In addition, in another embodiment, the expression vector contains aselectable marker gene to allow the selection of transformed host cells.Selection genes are well known in the art and will vary with the hostcell used.

The LIV-1 proteins of the present invention are produced by culturing ahost cell transformed with an expression vector containing nucleic acidencoding a LIV-1 protein, under the appropriate conditions to induce orcause expression of the LIV-1 protein. Conditions appropriate for LIV-1protein expression will vary with the choice of the expression vectorand the host cell, and will be easily ascertained by one skilled in theart through routine experimentation or optimization. For example, theuse of constitutive promoters in the expression vector will requireoptimizing the growth and proliferation of the host cell, while the useof an inducible promoter requires the appropriate growth conditions forinduction. In addition, in some embodiments, the timing of the harvestis important. For example, the baculoviral systems used in insect cellexpression are lytic viruses, and thus harvest time selection can becrucial for product yield.

Appropriate host cells include yeast, bacteria, archaebacteria, fungi,and insect and animal cells, including mammalian cells. Of particularinterest are Saccharomyces cerevisiae and other yeasts, E. coli,Bacillus subtilis, Sf9 cells, C129 cells, 293 cells, Neurospora, BHK,CHO, COS, HeLa cells, HUVEC (human umbilical vein endothelial cells),THP1 cells (a macrophage cell line) and various other human cells andcell lines.

In one embodiment, the LIV-1 proteins are expressed in mammalian cells.Mammalian expression systems are also known in the art, and includeretroviral and adenoviral systems. One expression vector system is aretroviral vector system such as is generally described inPCT/US97/01019 and PCT/US97/01048, both of which are hereby expresslyincorporated by reference. Of particular use as mammalian promoters arethe promoters from mammalian viral genes, since the viral genes areoften highly expressed and have a broad host range. Examples include theSV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirusmajor late promoter, herpes simplex virus promoter, and the CMV promoter(see, e.g., Fernandez & Hoeffler, supra). Typically, transcriptiontermination and polyadenylation sequences recognized by mammalian cellsare regulatory regions located 3′ to the translation stop codon andthus, together with the promoter elements, flank the coding sequence.Examples of transcription terminator and polyadenlyation signals includethose derived from SV40.

The methods of introducing exogenous nucleic acid into mammalian hosts,as well as other hosts, is well known in the art, and will vary with thehost cell used. Techniques include dextran-mediated transfection,calcium phosphate precipitation, polybrene mediated transfection,protoplast fusion, electroporation, viral infection, encapsulation ofthe polynucleotide(s) in liposomes, and direct microinjection of the DNAinto nuclei.

In some embodiments, LIV-1 proteins are expressed in bacterial systems.Bacterial expression systems are well known in the art. Promoters frombacteriophage may also be used and are known in the art. In addition,synthetic promoters and hybrid promoters are also useful; e.g., the tacpromoter is a hybrid of the trp and lac promoter sequences. Furthermore,a bacterial promoter can include naturally occurring promoters ofnon-bacterial origin that have the ability to bind bacterial RNApolymerase and initiate transcription. In addition to a functioningpromoter sequence, an efficient ribosome binding site is desirable. Theexpression vector may also include a signal peptide sequence thatprovides for secretion of the LIV-1 protein in bacteria. The protein iseither secreted into the growth media (gram-positive bacteria) or intothe periplasmic space, located between the inner and outer membrane ofthe cell (gram-negative bacteria). The bacterial expression vector mayalso include a selectable marker gene to allow for the selection ofbacterial strains that have been transformed. Suitable selection genesinclude genes which render the bacteria resistant to drugs such asampicillin, chloramphenicol, erythromycin, kanamycin, neomycin andtetracycline. Selectable markers also include biosynthetic genes, suchas those in the histidine, tryptophan and leucine biosynthetic pathways.These components are assembled into expression vectors. Expressionvectors for bacteria are well known in the art, and include vectors forBacillus subtilis, E. coli, Streptococcus cremoris, and Streptococcuslividans, among others. The bacterial expression vectors are transformedinto bacterial host cells using techniques well known in the art, suchas calcium chloride treatment, electroporation, and others.

In one embodiment, LIV-1 polypeptides are produced in insect cells.Expression vectors for the transformation of insect cells, and inparticular, baculovirus-based expression vectors, are well known in theart.

LIV-1 polypeptides can also be produced in yeast cells. Yeast expressionsystems are well known in the art, and include expression vectors forSaccharomyces cerevisiae, Candida albicans and C. maltosa, Hansenulapolymorpha, Kluyveromyces fragilis and K. lactis, Pichia guillerimondiiand P. pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica.

The LIV-1 polypeptides may also be made as a fusion protein, usingtechniques well known in the art. Thus, e.g., for the creation ofmonoclonal antibodies, if the desired epitope is small, the LIV-1protein may be fused to a carrier protein to form an immunogen.Alternatively, the LIV-1 protein may be made as a fusion protein toincrease expression, or for other reasons. For example, when the LIV-1protein is a LIV-1 peptide, the nucleic acid encoding the peptide may belinked to other nucleic acid for expression purposes.

The LIV-1 polypeptides are typically purified or isolated afterexpression. LIV-1 proteins may be isolated or purified in a variety ofways known to those skilled in the art depending on what othercomponents are present in the sample. Standard purification methodsinclude electrophoretic, molecular, immunological and chromatographictechniques, including ion exchange, hydrophobic, affinity, andreverse-phase HPLC chromatography, and chromatofocusing. For example,the LIV-1 protein may be purified using a standard anti-LIV-1 proteinantibody column. Ultrafiltration and diafiltration techniques, inconjunction with protein concentration, are also useful. For generalguidance in suitable purification techniques, see Scopes, ProteinPurification (1982). The degree of purification necessary will varydepending on the use of the LIV-1 protein. In some instances nopurification will be necessary.

One of skill will recognize that the expressed protein need not have thewild-type LIV-1 sequence but may be derivative or variant as compared tothe wild-type sequence. These variants typically fall into one or moreof three classes: substitutional, insertional or deletional variants.These variants ordinarily are prepared by site specific mutagenesis ofnucleotides in the DNA encoding the protein, using cassette or PCRmutagenesis or other techniques well known in the art, to produce DNAencoding the variant, and thereafter expressing the DNA in recombinantcell culture as outlined above. However, variant protein fragmentshaving up to about 100-150 residues may be prepared by in vitrosynthesis using established techniques Amino acid sequence variants arecharacterized by the predetermined nature of the variation, a featurethat sets them apart from naturally occurring allelic or interspeciesvariation of the LIV-1 protein amino acid sequence. The variantstypically exhibit the same qualitative biological activity as thenaturally occurring analogue, although variants can also be selectedwhich have modified characteristics as will be more fully outlinedbelow.

LIV-1 polypeptides of the present invention may also be modified in away to form chimeric molecules comprising a LIV-1 polypeptide fused toanother, heterologous polypeptide or amino acid sequence. In oneembodiment, such a chimeric molecule comprises a fusion of the LIV-1polypeptide with a tag polypeptide which provides an epitope to which ananti-tag antibody can selectively bind. The epitope tag is generallyplaced at the amino-or carboxyl-terminus of the LIV-1 polypeptide. Thepresence of such epitope-tagged forms of a LIV-1 polypeptide can bedetected using an antibody against the tag polypeptide. Also, provisionof the epitope tag enables the LIV-1 polypeptide to be readily purifiedby affinity purification using an anti-tag antibody or another type ofaffinity matrix that binds to the epitope tag. In an alternativeembodiment, the chimeric molecule may comprise a fusion of a LIV-1polypeptide with an immunoglobulin or a particular region of animmunoglobulin. For a bivalent form of the chimeric molecule, such afusion could be to the Fc region of an IgG molecule.

Various tag polypeptides and their respective antibodies are well knownin the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; HIS6 and metal chelationtags, the flu HA tag polypeptide and its antibody 12CA5 (Field et al.,Mol. Cell. Biol. 8:2159-2165 (1988)); the c-myc tag and the 8F9, 3C7,6E10, G4, B7 and 9E10 antibodies thereto (Evan et al., Molecular andCellular Biology 5:3610-3616 (1985)); and the Herpes Simplex virusglycoprotein D (gD) tag and its antibody (Paborsky et al., ProteinEngineering 3(6):547-553 (1990)). Other tag polypeptides include theFLAG-peptide (Hopp et al., BioTechnology 6:1204-1210 (1988)); the KT3epitope peptide (Martin et al., Science 255:192-194 (1992)); tubulinepitope peptide (Skinner et al., J. Biol. Chem. 266:15163-15166 (1991));and the T7 gene 10 protein peptide tag (Lutz-Freyermuth et al., Proc.Natl. Acad. Sci. USA 87:6393-6397 (1990)).

Antibodies to Cancer Proteins

Once the LIV-1 protein is produced, it is used to generate antibodies,e.g., for immunotherapy or immunodiagnosis. In some embodiments of theinvention, the antibodies recognize the same epitope as the CDRs shownin Table 2. The ability of a particular antibody to recognize the sameepitope as another antibody is typically determined by the ability ofone antibody to competitively inhibit binding of the second antibody tothe antigen. Any of a number of competitive binding assays can be usedto measure competition between two antibodies to the same antigen. Anexemplary assay is a BIACORE® (chemicals for use in biological assays)assay. Briefly in these assays, binding sites can be mapped instructural terms by testing the ability of interactants, e.g. differentantibodies, to inhibit the binding of another. Injecting two consecutiveantibody samples in sufficient concentration can identify pairs ofcompeting antibodies for the same binding epitope. The antibody samplesshould have the potential to reach a significant saturation with eachinjection. The net binding of the second antibody injection isindicative for binding epitope analysis. Two response levels can be usedto describe the boundaries of perfect competition versus non-competingbinding due to distinct epitopes. The relative amount of bindingresponse of the second antibody injection relative to the binding ofidentical and distinct binding epitopes determines the degree of epitopeoverlap.

Other conventional immunoassays known in the art can be used in thepresent invention. For example, antibodies can be differentiated by theepitope to which they bind using a sandwich ELISA assay. This is carriedout by using a capture antibody to coat the surface of a well. Asubsaturating concentration of tagged-antigen is then added to thecapture surface. This protein will be bound to the antibody through aspecific antibody:epitope interaction. After washing a second antibody,which has been covalently linked to a detectable moeity (e.g., HRP, withthe labeled antibody being defined as the detection antibody) is addedto the ELISA. If this antibody recognizes the same epitope as thecapture antibody it will be unable to bind to the target protein as thatparticular epitope will no longer be available for binding. If howeverthis second antibody recognizes a different epitope on the targetprotein it will be able to bind and this binding can be detected byquantifying the level of activity (and hence antibody bound) using arelevant substrate. The background is defined by using a single antibodyas both capture and detection antibody, whereas the maximal signal canbe established by capturing with an antigen specific antibody anddetecting with an antibody to the tag on the antigen. By using thebackground and maximal signals as references, antibodies can be assessedin a pair-wise manner to determine epitope specificity.

A first antibody is considered to competitively inhibit binding of asecond antibody, if binding of the second antibody to the antigen isreduced by at least 30%, usually at least about 40%, 50%, 60% or 75%,and often by at least about 90%, in the presence of the first antibodyusing any of the assays described above.

Methods of preparing polyclonal antibodies are known to the skilledartisan (e.g., Coligan, supra; and Harlow & Lane, supra). Polyclonalantibodies can be raised in a mammal, e.g., by one or more injections ofan immunizing agent and, if desired, an adjuvant. Typically, theimmunizing agent and/or adjuvant will be injected in the mammal bymultiple subcutaneous or intraperitoneal injections. The immunizingagent may include a protein encoded by a nucleic acid of the figures orfragment thereof or a fusion protein thereof. It may be useful toconjugate the immunizing agent to a protein known to be immunogenic inthe mammal being immunized. Examples of such immunogenic proteinsinclude but are not limited to keyhole limpet hemocyanin, serum albumin,bovine thyroglobulin, and soybean trypsin inhibitor. Examples ofadjuvants which may be employed include Freund's complete adjuvant andMPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalosedicorynomycolate). The immunization protocol may be selected by oneskilled in the art without undue experimentation.

The antibodies may, alternatively, be monoclonal antibodies. Monoclonalantibodies may be prepared using hybridoma methods, such as thosedescribed by Kohler & Milstein, Nature 256:495 (1975). In a hybridomamethod, a mouse, hamster, or other appropriate host animal, is typicallyimmunized with an immunizing agent to elicit lymphocytes that produce orare capable of producing antibodies that will specifically bind to theimmunizing agent. Alternatively, the lymphocytes may be immunized invitro. The immunizing agent will typically include a polypeptide encodedby a nucleic acid of Table 1, a fragment thereof, or a fusion proteinthereof. Generally, either peripheral blood lymphocytes (“PBLs”) areused if cells of human origin are desired, or spleen cells or lymph nodecells are used if non-human mammalian sources are desired. Thelymphocytes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(1986)). Immortalized cell lines are usually transformed mammaliancells, particularly myeloma cells of rodent, bovine and human origin.Usually, rat or mouse myeloma cell lines are employed. The hybridomacells may be cultured in a suitable culture medium that preferablycontains one or more substances that inhibit the growth or survival ofthe unfused, immortalized cells. For example, if the parental cells lackthe enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT orHPRT), the culture medium for the hybridomas typically will includehypoxanthine, aminopterin, and thymidine (“HAT medium”), whichsubstances prevent the growth of HGPRT-deficient cells.

In some embodiments the antibodies to the LIV-1 proteins are chimeric orhumanized antibodies. As noted above, humanized forms of antibodies arechimeric immunoglobulins in which residues from a complementarydetermining region (CDR) of human antibody are replaced by residues froma CDR of a non-human species such as mouse, rat or rabbit having thedesired specificity, affinity and capacity.

Human antibodies can be produced using various techniques known in theart, including phage display libraries (Hoogenboom & Winter, J. Mol.Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991)). Thetechniques of Cole et al. and Boerner et al. are also available for thepreparation of human monoclonal antibodies (Cole et al., MonoclonalAntibodies and Cancer Therapy, p. 77 (1985) and Boerner et al., J.Immunol. 147(1):86-95 (1991)). Similarly, human antibodies can be madeby introducing of human immunoglobulin loci into transgenic animals,e.g., mice in which the endogenous immunoglobulin genes have beenpartially or completely inactivated. Upon challenge, human antibodyproduction is observed, which closely resembles that seen in humans inall respects, including gene rearrangement, assembly, and antibodyrepertoire. This approach is described, e.g., in U.S. Pat. Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and inthe following scientific publications: Marks et al., Bio/Technology10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison,Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); Lonberg& Huszar, Intern. Rev. Immunol. 13:65-93 (1995).

In some embodiments, the antibody is a single chain Fv (scFv). The V_(H)and the V_(L) regions of a scFv antibody comprise a single chain whichis folded to create an antigen binding site similar to that found in twochain antibodies. Once folded, noncovalent interactions stabilize thesingle chain antibody. While the V_(H) and V_(L) regions of someantibody embodiments can be directly joined together, one of skill willappreciate that the regions may be separated by a peptide linkerconsisting of one or more amino acids. Peptide linkers and their use arewell-known in the art. See, e.g., Huston et al., Proc. Nat'l Acad. Sci.USA 8:5879 (1988); Bird et al., Science 242:4236 (1988); Glockshuber etal., Biochemistry 29:1362 (1990); U.S. Pat. No. 4,946,778, U.S. Pat. No.5,132,405 and Stemmer et al., Biotechniques 14:256-265 (1993). Generallythe peptide linker will have no specific biological activity other thanto join the regions or to preserve some minimum distance or otherspatial relationship between the V_(H) and V_(L). However, theconstituent amino acids of the peptide linker may be selected toinfluence some property of the molecule such as the folding, net charge,or hydrophobicity. Single chain Fv (scFv) antibodies optionally includea peptide linker of no more than 50 amino acids, generally no more than40 amino acids, preferably no more than 30 amino acids, and morepreferably no more than 20 amino acids in length. In some embodiments,the peptide linker is a concatamer of the sequence Gly-Gly-Gly-Gly-Ser,preferably 2, 3, 4, 5, or 6 such sequences. However, it is to beappreciated that some amino acid substitutions within the linker can bemade. For example, a valine can be substituted for a glycine.

Methods of making scFv antibodies have been described. See, Huse et al.,supra; Ward et al. supra; and Vaughan et al., supra. In brief, mRNA fromB-cells from an immunized animal is isolated and cDNA is prepared. ThecDNA is amplified using primers specific for the variable regions ofheavy and light chains of immunoglobulins. The PCR products are purifiedand the nucleic acid sequences are joined. If a linker peptide isdesired, nucleic acid sequences that encode the peptide are insertedbetween the heavy and light chain nucleic acid sequences. The nucleicacid which encodes the scFv is inserted into a vector and expressed inthe appropriate host cell. The scFv that specifically bind to thedesired antigen are typically found by panning of a phage displaylibrary. Panning can be performed by any of several methods. Panning canconveniently be performed using cells expressing the desired antigen ontheir surface or using a solid surface coated with the desired antigen.Conveniently, the surface can be a magnetic bead. The unbound phage arewashed off the solid surface and the bound phage are eluted.

Finding the antibody with the highest affinity is dictated by theefficiency of the selection process and depends on the number of clonesthat can be screened and the stringency with which it is done.Typically, higher stringency corresponds to more selective panning Ifthe conditions are too stringent, however, the phage will not bind.After one round of panning, the phage that binds to LIV-1 coated platesor to cells expressing LIV-1 on their surface are expanded in E. coliand subjected to another round of panning. In this way, an enrichment ofmany fold occurs in 3 rounds of panning. Thus, even when enrichment ineach round is low, multiple rounds of panning will lead to the isolationof rare phage and the genetic material contained within which encodesthe scFv with the highest affinity or one which is better expressed onphage.

Regardless of the method of panning chosen, the physical link betweengenotype and phenotype provided by phage display makes it possible totest every member of a cDNA library for binding to antigen, even withlarge libraries of clones.

In one embodiment, the antibodies are bispecific antibodies. Bispecificantibodies are monoclonal, preferably human or humanized, antibodiesthat have binding specificities for at least two different antigens orthat have binding specificities for two epitopes on the same antigen. Inone embodiment, one of the binding specificities is for the LIV-1protein, the other one is for another cancer antigen. Alternatively,tetramer-type technology may create multivalent reagents.

In some embodiments, the antibodies to LIV-1 protein are capable ofreducing or eliminating cells expressing LIV-1 (e.g., prostate cancer orbreast cancer cells). Generally, at least a 25% decrease in activity,growth, size or the like is preferred, with at least about 50% beingparticularly preferred and about a 95-100% decrease being especiallypreferred.

By immunotherapy is meant treatment of prostate cancer or breast cancerwith an antibody raised against LIV-1 proteins. As used herein,immunotherapy can be passive or active. Passive immunotherapy as definedherein is the passive transfer of antibody to a recipient (patient).Active immunization is the induction of antibody and/or T-cell responsesin a recipient (patient). Induction of an immune response is the resultof providing the recipient with an antigen (e.g., LIV-1 or DNA encodingit) to which antibodies are raised. As appreciated by one of ordinaryskill in the art, the antigen may be provided by injecting a polypeptideagainst which antibodies are desired to be raised into a recipient, orcontacting the recipient with a nucleic acid capable of expressing theantigen and under conditions for expression of the antigen, leading toan immune response.

In some embodiments, the antibody is conjugated to an effector moiety.The effector moiety can be any number of molecules, including labelingmoieties such as radioactive labels or fluorescent labels, or can be atherapeutic moiety. In one aspect the therapeutic moiety is a smallmolecule that modulates the activity of the LIV-1 protein. In anotheraspect the therapeutic moiety modulates the activity of moleculesassociated with or in close proximity to the LIV-1 protein.

In other embodiments, the therapeutic moiety is a cytotoxic agent. Inthis method, targeting the cytotoxic agent to prostate cancer or breastcancer tissue or cells, results in a reduction in the number ofafflicted cells, thereby reducing symptoms associated with prostatecancer or breast cancer. Cytotoxic agents are numerous and varied andinclude, but are not limited to, cytotoxic drugs or toxins or activefragments of such toxins. Suitable toxins and their correspondingfragments include diphtheria A chain, exotoxin A chain, ricin A chain,abrin A chain, curcin, crotin, phenomycin, enomycin, auristatin-E andthe like. Cytotoxic agents also include radiochemicals made byconjugating radioisotopes to antibodies raised against prostate canceror breast cancer proteins, or binding of a radionuclide to a chelatingagent that has been covalently attached to the antibody. Targeting thetherapeutic moiety to transmembrane prostate cancer or breast cancerproteins not only serves to increase the local concentration oftherapeutic moiety in the prostate cancer or breast cancer afflictedarea, but also serves to reduce deleterious side effects that may beassociated with the therapeutic moiety.

Binding Affinity of Antibodies of the Invention

Binding affinity for a target antigen is typically measured ordetermined by standard antibody-antigen assays, such as BIACORE®competitive assays, saturation assays, or immunoassays such as ELISA orRIA.

Such assays can be used to determine the dissociation constant of theantibody. The phrase “dissociation constant” refers to the affinity ofan antibody for an antigen. Specificity of binding between an antibodyand an antigen exists if the dissociation constant (K_(D)=1/K, where Kis the affinity constant) of the antibody is <1 μM, preferably <100 nM,and most preferably <0.1 nM. Antibody molecules will typically have aK_(D) in the lower ranges. K_(D)=[Ab−Ag]/[Ab] [Ag] where [Ab] is theconcentration at equilibrium of the antibody, [Ag] is the concentrationat equilibrium of the antigen and [Ab−Ag] is the concentration atequilibrium of the antibody-antigen complex. Typically, the bindinginteractions between antigen and antibody include reversible noncovalentassociations such as electrostatic attraction, Van der Waals forces andhydrogen bonds.

The antibodies of the invention specifically bind to LIV-1 proteins. By“specifically bind” herein is meant that the antibodies bind to theprotein with a K_(D) of at least about 0.1 mM, more usually at leastabout 1 μM, preferably at least about 0.1 μM or better, and mostpreferably, 0.01 μM or better.

Immunoassays

The antibodies of the invention can be used to detect LIV-1 or LIV-1expressing cells using any of a number of well recognized immunologicalbinding assays (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110;4,517,288; and 4,837,168). For a review of the general immunoassays, seealso Methods in Cell Biology, Vol. 37, Asai, ed. Academic Press, Inc.New York (1993); Basic and Clinical Immunology 7th Edition, Stites &Terr, eds. (1991).

Thus, the present invention provides methods of detecting cells thatexpress LIV-1. In one method, a biopsy is performed on the subject andthe collected tissue is tested in vitro. The tissue or cells from thetissue is then contacted, with an anti-LIV-1 antibody of the invention.Any immune complexes which result indicate the presence of a LIV-1protein in the biopsied sample. To facilitate such detection, theantibody can be radiolabeled or coupled to an effector molecule which isa detectable label, such as a radiolabel. In another method, the cellscan be detected in vivo using typical imaging systems. Then, thelocalization of the label is determined by any of the known methods fordetecting the label. A conventional method for visualizing diagnosticimaging can be used. For example, paramagnetic isotopes can be used forMRI. Internalization of the antibody may be important to extend the lifewithin the organism beyond that provided by extracellular binding, whichwill be susceptible to clearance by the extracellular enzymaticenvironment coupled with circulatory clearance.

LIV-1 proteins can also be detected using standard immunoassay methodsand the antibodies of the invention. Standard methods include, forexample, radioimmunoassay, sandwich immunoassays (including ELISA),immunofluorescence assays, Western blot, affinity chromatography(affinity ligand bound to a solid phase), and in situ detection withlabeled antibodies.

Suppression of Endogenous LIV-1 Gene Expression Through the Use of RNAi

In many species, introduction of double-stranded RNA (dsRNA) which mayalternatively be referred to herein as small interfering RNA (siRNA),induces potent and specific gene silencing, a phenomena called RNAinterference or RNAi. siRNA, in particular, is capable of renderinggenes nonfunctional in a sequence specific manner. This phenomenon hasbeen extensively documented in the nematode C. elegans (Fire, A., et al,Nature, 391, 806-811, 1998), but is widespread in other organisms,ranging from trypanasomes to mouse. Recent experiments demonstrate theinhibition of gene expression in human somatic cells, including theembryonic kidney cell line 293 and the epithelial carcinoma cell lineHeLa. Caplen, N. J., et al., P.N.A.S. 98:9742-9747 (2001). Depending onthe organism being discussed, RNA interference has been referred to as“co-suppression”, “post-transcriptional gene silencing”, “sensesuppression” and “quelling”.

RNAi is attractive as a biotechnological tool because it provides ameans for knocking out the activity of specific genes. It isparticularly useful for knocking out gene expression in species thatwere not previously considered to be amenable to genetic analysis ormanipulation.

In designing RNAi experiments there are several factors that need to beconsidered such as the nature of the dsRNA, the durability of thesilencing effect, and the choice of delivery system. See Elbashir, S. M.et al., EMBO J. 20:6877-6888 (2001).

To produce an RNAi effect, the dsRNA, or siRNA that is introduced intothe organism should contain exonic sequences. Furthermore, the RNAiprocess is homology dependent, so the sequences must be carefullyselected so as to maximize gene specificity, while minimizing thepossibility of cross-interference between homologous, but notgene-specific sequences. Preferably the dsRNA exhibits greater than 90%or even 100% identity between the sequence of the dsRNA and the gene tobe inhibited. Sequences less than about 80% identical to the target geneare substantially less effective. Thus, the greater homology between thedsRNA and the gene whose expression is to be inhibited, the less likelyexpression of unrelated genes will be affected.

In addition, the size of the dsRNA is important. dsRNA may be greaterthan 500 base pairs in length, however, smaller fragments can alsoproduce an RNAi effect. In particular, fragments that are short enoughto avoid activation of nonsequence specific dsRNA responses (e.g.interferon responses) are effective in silencing gene responses. SeeElbashir, S. M., et al., Nature 411:494-498 (2001).

Introduction of dsRNA can be achieved by any method known in the art,including for example, microinjection, liposome transfection orelectroporation. A variety of mechanisms by which dsRNA may inhibit geneexpression have been proposed, but evidence in support of any specificmechanism is lacking (Fire, A., 1999; Caplen, N. J. et al., 2001).

Administration of Pharmaceutical and Vaccine Compositions

The antibodies, nucleic acids and polypeptides of the invention can beformulated in pharmaceutical compositions. Thus, the invention alsoprovide methods and compositions for administering a therapeuticallyeffective dose of an anti-LIV-1 antibody, a LIV-1 nucleic acid, or aLIV-1 polypeptide or protein. The exact dose will depend on the purposeof the treatment, and will be ascertainable by one skilled in the artusing known techniques (e.g., Ansel et al., Pharmaceutical Dosage Formsand Drug Delivery; Lieberman, Pharmaceutical Dosage Forms (vols. 1-3,1992), Dekker, ISBN 0824770846, 082476918X, 0824712692, 0824716981;Lloyd, The Art, Science and Technology of Pharmaceutical Compounding(1999); and Pickar, Dosage Calculations (1999)). As is known in the art,adjustments for prostate cancer or breast cancer degradation, systemicversus localized delivery, and rate of new protease synthesis, as wellas the age, body weight, general health, sex, diet, time ofadministration, drug interaction and the severity of the condition maybe necessary, and will be ascertainable with routine experimentation bythose skilled in the art.

A “patient” for the purposes of the present invention includes bothhumans and other animals, particularly mammals. Thus the methods areapplicable to both human therapy and veterinary applications. In oneembodiment the patient is a mammal, preferably a primate. In otherembodiments the patient is human.

The administration of the antibodies, nucleic acids and polypeptides ofthe present invention can be done in a variety of ways as discussedabove, including, but not limited to, orally, subcutaneously,intravenously, intranasally, transdermally, intraperitoneally,intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly.

The pharmaceutical compositions of the present invention comprise anantibody, nucleic acid or polypeptide of the invention in a formsuitable for administration to a patient. In one embodiment, thepharmaceutical compositions are in a water soluble form, such as beingpresent as pharmaceutically acceptable salts, which is meant to includeboth acid and base addition salts. “Pharmaceutically acceptable acidaddition salt” refers to those salts that retain the biologicaleffectiveness of the free bases and that are not biologically orotherwise undesirable, formed with inorganic acids such as hydrochloricacid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid andthe like, and organic acids such as acetic acid, propionic acid,glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid,succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid,cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid and the like. “Pharmaceuticallyacceptable base addition salts” include those derived from inorganicbases such as sodium, potassium, lithium, ammonium, calcium, magnesium,iron, zinc, copper, manganese, aluminum salts and the like. Particularlyuseful are the ammonium, potassium, sodium, calcium, and magnesiumsalts. Salts derived from pharmaceutically acceptable organic non-toxicbases include salts of primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as isopropylamine,trimethylamine, diethylamine, triethylamine, tripropylamine, andethanolamine.

The pharmaceutical compositions may also include one or more of thefollowing: carrier proteins such as serum albumin; buffers; fillers suchas microcrystalline cellulose, lactose, corn and other starches; bindingagents; sweeteners and other flavoring agents; coloring agents; andpolyethylene glycol.

The pharmaceutical compositions can be administered in a variety of unitdosage forms depending upon the method of administration. For example,unit dosage forms suitable for oral administration include, but are notlimited to, powder, tablets, pills, capsules and lozenges. It isrecognized that compositions of the invention when administered orally,should be protected from digestion. This is typically accomplishedeither by complexing the molecules with a composition to render themresistant to acidic and enzymatic hydrolysis, or by packaging themolecules in an appropriately resistant carrier, such as a liposome or aprotection barrier. Means of protecting agents from digestion are wellknown in the art.

The compositions for administration will commonly comprise an antibody,polypeptide or nucleic acid of the invention dissolved in apharmaceutically acceptable carrier, preferably an aqueous carrier. Avariety of aqueous carriers can be used, e.g., buffered saline and thelike. These solutions are sterile and generally free of undesirablematter. These compositions may be sterilized by conventional, well knownsterilization techniques. The compositions may contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions such as pH adjusting and buffering agents, toxicity adjustingagents and the like, e.g., sodium acetate, sodium chloride, potassiumchloride, calcium chloride, sodium lactate and the like. Theconcentration of active agent in these formulations can vary widely, andwill be selected primarily based on fluid volumes, viscosities, bodyweight and the like in accordance with the particular mode ofadministration selected and the patient's needs (e.g., Remington'sPharmaceutical Science (15th ed., 1980) and Goodman & Gillman, ThePharmacologial Basis of Therapeutics (Hardman et al.,eds., 1996)).

Thus, a typical pharmaceutical composition for intravenousadministration would be about 0.1 to 10 mg per patient per day. Dosagesfrom 0.1 up to about 100 mg per patient per day may be used,particularly when the drug is administered to a secluded site and notinto the blood stream, such as into a body cavity or into a lumen of anorgan. Substantially higher dosages are possible in topicaladministration. Actual methods for preparing parenterally administrablecompositions will be known or apparent to those skilled in the art,e.g., Remington's Pharmaceutical Science and Goodman and Gillman, ThePharmacologial Basis of Therapeutics, supra.

The compositions containing antibodies, polypeptides or nucleic acids ofthe invention can be administered for therapeutic or prophylactictreatments. In therapeutic applications, compositions are administeredto a patient suffering from a disease (e.g., a cancer) in an amountsufficient to cure or at least partially arrest the disease and itscomplications. An amount adequate to accomplish this is defined as a“therapeutically effective dose.” Amounts effective for this use willdepend upon the severity of the disease and the general state of thepatient's health. Single or multiple administrations of the compositionsmay be administered depending on the dosage and frequency as requiredand tolerated by the patient. In any event, the composition shouldprovide a sufficient quantity of the agents of this invention toeffectively treat the patient. An amount of modulator that is capable ofpreventing or slowing the development of cancer in a mammal is referredto as a “prophylactically effective dose.” The particular dose requiredfor a prophylactic treatment will depend upon the medical condition andhistory of the mammal, the particular cancer being prevented, as well asother factors such as age, weight, gender, administration route,efficiency, etc. Such prophylactic treatments may be used, e.g., in amammal who has previously had cancer to prevent a recurrence of thecancer, or in a mammal who is suspected of having a significantlikelihood of developing cancer.

It will be appreciated that the present prostate cancer or breast cancerprotein-modulating compounds can be administered alone or in combinationwith additional prostate cancer or breast cancer modulating compounds orwith other therapeutic agent, e.g., other anti-cancer agents ortreatments.

Kits for Use in Diagnostic and/or Prognostic Applications

For use in diagnostic, research, and therapeutic applications suggestedabove, kits are also provided by the invention. In the diagnostic andresearch applications such kits may include any or all of the following:assay reagents, buffers, and LIV-1-specific antibodies of the invention.A therapeutic product may include sterile saline or anotherpharmaceutically acceptable emulsion and suspension base.

In addition, the kits may include instructional materials containingdirections (i.e., protocols) for the practice of the methods of thisinvention. While the instructional materials typically comprise writtenor printed materials they are not limited to such. Any medium capable ofstoring such instructions and communicating them to an end user iscontemplated by this invention. Such media include, but are not limitedto electronic storage media (e.g., magnetic discs, tapes, cartridges,chips), optical media (e.g., CD ROM), and the like. Such media mayinclude addresses to internet sites that provide such instructionalmaterials.

EXAMPLES Example 1 Antibodies to the Target Protein LIV-1, InhibitProstate Tumor Cell Growth In Vivo

The following example illustrates that LIV-1 antibodies are effective atreducing tumor volume in vivo. Animal studies were conducted using maleSCID mice implanted with a prostate cancer cell line, LNCaP. The LNCaPcell line expresses the antigen recognized by LIV-1 antibodies. Theprotein and nucleic acid sequences of the anti-LIV-1 #1.7A4 antibodieswhich were effective in these experiments, are provided as SEQ ID NOS:3, 4, 5, and 6 (Table 2). Tumors were allowed to grow until they reacheda size of between 50-100 mm³. At that time, animals were randomized intogroups and subjected to treatment with either a.) anAuristatin-E-conjugated isotype control antibody, or b.) theAuristatin-E-conjugated LIV-1 antibody, 1.7A4.

Antibodies were administered at a dose of 10 mg/kg for a total of 10doses given intra-peritoneally every four days. Tumor size was measuredtwice weekly for 38 days. At the conclusion of the study, only tumors inthe group treated with Auristatin-E-conjugated LIV-1 antibodies showedregression. See FIG. 1.

Thus, these experiments showed that treatment with theAuristatin-E-conjugated LIV-1 antibody results in a significant tumorvolume reduction. Therefore the Auristatin-E-conjugated LIV-1 antibodiesfunction as anti-cancer therapeutics for the treatment of patientsbearing LIV-1 expressing tumors.

Example 2 Antibodies to the Target Protein LIV-1, Inhibit Breast TumorCell Growth in Vivo

The following example illustrates that LIV-1 antibodies are effective atreducing tumor volume in vivo. Animal studies were conducted usingfemale SCID mice implanted with estrogen pellets. The fat pads of themice were implanted with a breast cancer cell line, MCF7. The MCF7 cellline expresses the antigen recognized by LIV-1 antibodies. The proteinand nucleic acid sequences of the anti-LIV-1 #1.7A4 antibodies whichwere one of the auristatin-E-conjugated-LIV-1 antibodies effective inthese experiments, are provided as SEQ ID NOs: 3, 4, 5, and 6 (Table 2).

Tumors were allowed to grow until they reached a size of between 50-100mm³. At that time, animals were randomized into groups and subjected totreatment with either a.) vehicle, b.) an Auristatin-E-conjugatedisotype control antibody, or c.) one of two Auristatin-E-conjugatedLIV-1 antibodies, 1.7A4 or 1.1E10.

Antibodies were administered at a dose of 5 mg/kg for a total of 5 dosesgiven intra-peritoneally every four days. Tumor size was measure twiceweekly for 30 days. At the conclusion of the study, only tumors in thegroup treated with Auristatin-E-conjugated LIV-1 antibodies showedregression. See FIG. 2.

Thus, these experiments showed that treatment with theAuristatin-E-conjugated LIV-1 antibody results in a significant tumorvolume reduction. Therefore the Auristatin-E-conjugated LIV-1 antibodiesfunction as anti-cancer therapeutics for the treatment of patientsbearing LIV-1 expressing tumors.

Example 3 Immunohistochemistry Analysis Using LIV-1 Antibodies

Tissue microarrays of primary breast cancer samples were obtained fromClinomics Biosciences, Inc. (Pittsfield, Mass.). Normal body tissuesspecimens were collected from samples harvested at the time of cadavericorgan donation from 6 individuals (3 males, 3 females obtained fromZoion, Hawthorne, N.Y.). IHC on formalin-fixed paraffin embedded tissueswas performed using standard methods: heat induced antigen retrieval wasperformed in Dako Target Retrieval Solution for 15 minutes in a pressurecooker. Samples were then incubated with 1.1F10 anti-LIV-1 antibodies orcontrol mouse IgG1 [TIB191, a mouse anti-trinitrophenol mAb (hybridomaclone 1B76.11)] for 30 minutes. Antibody binding was detected usingbiotinylated secondary antibody [Goat-anti-mouse IgG (3 mg/ml, 30minutes; Jackson ImmunoResearch)], and developed using the VectastainElite ABC Kit (Vector Laboratories) and stable DAB (diaminobenzidine andH2O2; Research Genetics). Staining was performed using the DAKOAutostainer at room temperature.

Analysis of primary breast cancer specimens showed that LIV-1-specificstaining was restricted to the cytoplasm and membranes of the breastcancer epithelium, as compared to tissue specimens from pancreas, liver,skeletal muscle, adrenal gland, heart, spleen, cerebellum, lung,duodenum, kidney, myometrium and placenta, which showed no significantstaining. See FIG. 3. The breast cancer cohort (n=133) displayed weak tostrong staining in 27% of the cases, demonstrating that a large fractionof breast cancer patients exhibit expression of LIV-1. Analysis of aprostate cancer cohort (87 cases) demonstrated significant LIV-1expression in 42% of the cases (data not shown). The staining in theprostate cancer specimens was also restricted to the glandularepithelium. These results demonstrate that LIV-1 protein is highlyexpressed in both breast and prostate cancer specimens. Therefore, LIV-1is an attractive target for antibody-based therapy for both breast andprostate cancers.

Example 4 LIV-1 RNAi Clonogenic Assay

An RNAi Clonogenic assay was performed to determine the extent of LIV-1siRNA targeted inhibition of carcinoma cell proliferation. MX-1 breastcarcinoma cells were transfected with siRNAs using Lipofectamine 2000(Invitrogen) according to the manufacturer's instructions except thatcells were transfected in suspension as follows: MX-1 cells were dilutedto 500 cells/ml. 0.5 ml was placed into each well of a 24 well platecontaining 100 ul of a mixture of Lipofectamine 2000 (Invitrogen) andsiRNAs in Minimal Essential Medium without phenol red (Invitrogen), witha final siRNA concentration of 10 nM. Each siRNA was assayed in fourreplicate wells. Cells were incubated at 37 C. for 14-17 days, duringwhich time the media was changed twice a week. The extent of cellproliferation was assessed by adding alamar blue to a finalconcentration of 12 ug/ml for 4-8 hours. Fluorescence was measured byexcitation at 544 nm and emission at 590 nm.

siRNAs were purchased from Dharmacon as duplexes with 3′ dTdT overhangs.siRNA sequences used are as follows:

H2R-1 (negative control) sense (SEQ ID NO: 7):5′-CAGACACGGCCACGUGUGAdTdT-3′ H2R-1 antisense (SEQ ID NO: 8):5′-UCACACGUGGCCGUGUCUGdTdT-3′HKSP-1 (positive control) sense (SEQ ID NO: 9):5′-GCUAGCGCCCAUUCAAUAGdTdT-3′ HKSP-1 antisense (SEQ ID NO: 10):5′-CUAUUGAAUGGGCGCUAGCdTdT-3′ BCR4-53 sense (SEQ ID NO: 11):5′-CAGCUUUUCUACCGCUAUGdTdT-3′ BCR4-53 antisense (SEQ ID NO: 12):5′-CAUAGCGGUAGAAAAGCUGdTdT-3′

The results indicate that downmodulation of LIV-1 by siRNA reducedcarcinoma cell proliferation, as compared to controls. This indicatesthat LIV-1 expression is essential for proliferation in these carcinomacells. As seen in FIGS. 4A & 4B, LIV-1 siRNA (BCR4-53; SEQ ID NOS:11 and12) decreased cellular proliferation as compared to a non-specific siRNAcontrol (H2R-1; SEQ ID NOS:7 and 8) in both 14- and 17-day culturesafter addition of siRNA. The inhibition of cellular proliferation byLIV-1 siRNA was also comparable to a known inhibitor of carcinoma cellproliferation, HKSP-1 (SEQ ID NOS:9 and 10). This experiment, therefore,validates LIV-1 as a legitimate target for inhibiting cellproliferation.

Example 5 MMP Activity Assay

LIV-1 siRNA was also tested in a matrix metalloproteinase (MMP) activityassay. Cancer cells exhibit a variety of MMP activity, including MMP-2and MMP-9 gelatinase activity. Increased MMP activity has been directlyimplicated in tumor cell invasion and angiogenesis because of theirability to degrade extracellular matrix components (see Olson, M. W. etal., J. Biol. Chem. 272:29975 (1997); MacDougall, J. R. et al., CancerMetastasis Rev. 14:351 (1995); Cockett, M. I. et al, Biochem. Soc. Symp.63:295 (1998)). MMP-2 and MMP-9 activity can be measured by determiningthe extent of gelatin degradation upon addition of carcinoma cells ontoa gelatin matrix.

HCT-116 colorectal adenocarcinoma cells (obtained from NCI) weretransfected with either BCR4-53 or H2R-1 siRNAs as described in the RNAiclonogenic assay with the following changes: 2.5 ml of cells at 2×10⁴cells/ml were plated into a single well of a 6 well plate containing 5ml Lipofectamine-2000/siRNA mixture for a final siRNA concentration of10 nM.

Cells were harvested approximately 72 hours after transfection bytrypsinization, and counted using a hemacytometer. 1×10⁴ cells in 1 mlmedia were plated onto fluorescent gelatin-coated glass coverslips in 24well plates. Coverslips were prepared by acid washing and coated with 10ul 0.5 mg/ml gelatin conjugated to Oregon Green (Molecular Probes).After drying, the gelatin was fixed with 0.5% glutaraldehyde for 15minutes on ice, washed several times with H2O, and sterilized with 70%ethanol before the cells were plated on top. The plates were incubatedat 37 C. to allow the cells to attach and degrade the gelatin.

Approximately 24 hours after plating, the cells were fixed inCytofix/Cytoperm (BD-Pharmingen). Total cellular LIV-1 protein levelswere then assessed with anti-LIV-1 antibody (1.7A4) at 10 ug/ml,followed by F(ab′)₂ goat anti-mouse IgG conjugated to Alexa Fluor 594(Molecular Probes). Cell nuclei were also stained with 10 uM Hoechst33342. The coverslips were mounted onto glass slides and subjected tofluorescence microscopy.

The results show (compare FIGS. 5A & 5B) that LIV-1 siRNA (SEQ ID NOS:11 and 12) inhibited gelatin degradation by HCT-116 colorectaladenocarcinoma cells. This indicates, that LIV-1 is important in theupregulation of MMP—expression and/or activity in HCT-116 colorectaladenocarcinoma cells, and therefore validates LIV-1's role as a mediatorin the extracellular invasion, and extracellular invasion andangiogenesis upregulation by carcinoma cells.

Example 6 Additional Antibodies to LIV-1

Monoclonal antibodies were raised against an N-terminus LIV-1 antigenand tested for binding on MX-1 cells by titration through FACS analysis.Hybridomas were derived from a fusion of NSO mouse myeloma cells andlymph nodes from mice immunized with purified protein containing theN-terminal 329 aa of LIV-1 fused to the human Fc domain. Hybridomaswhich expressed antibodies specific for LIV-1 were subcloned, implantedinto mice, and antibodies were purified from ascites fluid.

Comparative binding of the various LIV-1 antibodies was assessed by flowcytometry. MX-1 breast carcinoma cells were incubated with a dilutionseries of LIV-1 and control antibody, followed by incubation with goatanti-mouse IgG conjugated to FITC (Caltag). Each dilution was done intriplicate.

The FACS results in FIG. 6 indicate that several LIV-1 antibodies showpromising binding levels on MX-1 cells. In particular, LIV-1 antibodies14 (ATCC accession number PTA-5705), 19 (ATCC accession number PTA-5706and 23 (ATCC accession number PTA-5707).

Example 7 In Vitro Proliferation Assay with Toxin-Conjugated LIV-1Antibodies

LIV-1 and control antibodies were conjugated to Auristatin-E containinga valine-citrulline peptide linker and purified by HPLC. Serialdilutions of two LIV-1 antibodies, 14a and 22a, were compared to apreviously tested LIV-1 antibody (see Examples 1 and 2, supra), 1.7A4,and an IgG isotype control antibody for their ability to be internalizedand kill LIV-1 expressing cells. 50 ul cells were plated into 96 wellplates: CSOC 882-2 ovarian carcinoma cells at 650 cells/well, LNCaPprostate adenocarcinoma cells at 5000 cells/well, and MX-1 breastcarcinoma cells at 650 cells/well. 24 hours after plating, 50 ul of theappropriate antibody dilution was added to triplicate wells andincubated for an additional 72 or 96 hours at 37 C. The extent of cellproliferation was assessed by adding alamar blue to a finalconcentration of 12 ug/ml and incubating at 37 C. for 2 hours.Fluorescence was measured by excitation at 544 nm and emission at 590nm. Fraction survival was calculated by normalizing cell survival ofantibody-exposed cells to control cells growth in the absence ofantibody.

The results in FIGS. 7, 8 and 9 indicate that auristatin-E-conjugated14a and 22a LIV-1 antibodies were more effective in in vitro growthassays using a variety of carcinoma cell types than theauristatin-E-conjugated 1.7a4 LIV-1 antibody. For CSOC (FIG. 7), MX-1(FIG. 8) and LNCaP cells (FIG. 9), both 14a and 22a conjugatedantibodies were more effective at tumor cell growth suppression than1.7A4 LIV-1 antibody and a control IgG1 antibody. Therefore, the LIV-114a and 22a antibodies are useful in tumor cell growth suppression.

Example 8 Epitope Mapping of Anti-LIV-1 Antibodies Identifies ThreeDistinct Epitopes

Epitope mapping of BCR4 antibodies was done with flow cytometry using acompetitive binding assay. MX-1 breast carcinoma cells were incubatedwith one of three FITC-conjugated antibodies in the presence or absenceof a 10-fold molar excess of unconjugated (or naked) antibodies. In someexperiments, the naked antibody was used at 5- or 20-fold molar excesswith similar results. The ability of each naked antibody to compete witha FITC-conjugated antibody for binding was assessed. In all cases, nakedantibodies either did not compete at all (−) or competed as well as thecognate naked antibody (+).

FITC-conjugated Abs Naked Abs 14 19 21 13 − + − 14 + − + 15 + ND + 16 +ND + 19 − + − 20 + ND + 21 + ND + 22 + ND + 23 + ND + 24 + ND + 25 +ND + 1.7A4 − + − 1.1F10 − − − IGG1 − − − ISOTYPE ND: not done

From this experiment we demonstrate that 9 of the antibodies bind thesame or overlapping epitopes. These antibodies are 14, 15, 16, 20, 21,22, 23, 24, and 25. The epitope bound by these antibodies is distinctfrom those of the previous antibodies we had generated and thusrepresents a novel epitope for potential therapeutic applications. Theother two new antibodies, 13 and 19, represent an epitope binding groupcontaining one of our previous antibodies, 1.7A4. A third epitopebinding group, represented by 1.1F10 from our previous set ofantibodies, was not represented in this new panel of antibodies.

Example 9 Mutant LIV-1 Protein

A mutant LIV-1 protein (BCR4M1 cDNA (SEQ ID NO:13) and protein sequence(SEQ ID NO:14) provided in Table 5) was generated with a mutation in theputative MMP/Zn transporter domain. The goal to generating this mutantwas to further characterize the biological activity of LIV-1.

This mutant was made using two rounds of PCR. Briefly two fragments weregenerated from the wild type gene via PCR using internal primerscarrying the desired mutations.

The 5′ end was generated using primers

(SEQ ID NO: 15) 1 (CTTTAATTAACACCGCCACCATGGCGAGGAAGTTATCTGTAATC) and(SEQ ID NO: 16) 2 (TAATGCAGCAGGCAACGCAGCACAGAACACAGCAACAGAAG).

The 3′ end was generated using primers

(SEQ ID NO: 17) 3 (TGCTGCGTTGCCTGCTGCATTAGGTGACTTTGCTGTTC) and (SEQ IDNO: 18) 4 (GTCTCGAGGAAATTTATACGAAAC).

Since primers 2 and 3 contain overlapping sequences the two genefragments were gel purified and used as overlapping templates in asecond round of PCR using primers 1 and 4. The product of this secondround PCR were cloned into pCR4 topo blunt and selected clones weresequenced. The desired mutant was selected and subcloned into the NEF39expression vector and transfected into 3T12 cells. Overexpressing cellswere isolated using a CD4-based screening procedure.

The isolated clones expressed significantly higher levels of mutantLIV-1 protein compared to clones isolated from experiments in whichwild-type LIV-1 protein were transfected into 3T12 cells (data notshown). This result suggests that a functional activity, possibly MMPand/or Zn transporter activity, limits the amount of LIV-1 protein thatcan be expressed in a cell. It is known that expressing high levels ofcertain oncogenes in cells is difficult, because these oncogenesactivate signaling pathways that under normal tissue culture conditionslead to cell death. It is possible that LIV-1 activity has a similareffect in cells and that mutational inactivation abrogates this effect,allowing for higher overexpression. This result suggests a functionalrole for LIV-1 in regulating cell proliferation and/or cell survival.

Example 10 Use of LIV-1 Antibodies to Delay the Onset ofAndrogen-Independence of Prostate Cancer and/or to TreatAndrogen-Independent Disease

Prostate cancer is a hormone regulated disease that affects men in thelater years of life. Untreated prostate cancer metastasizes to lymphnodes and bone in advanced cases. In such cases current treatmentconsists of antagonizing the androgenic growth-stimulus that feeds thetumor by chemical or surgical hormone-ablation therapy (Galbraith andDuchesne. (1997) Eur. J. Cancer 33:545-554). An unfortunate consequenceof anti-androgen treatment is the development of androgen-independentcancer. Androgen regulated genes such as the gene encodingprostate-specific antigen (PSA) are turned off with hormone-ablationtherapy, but reappear when the tumor becomes androgen-independent(Akakura et al. (1993) Cancer 71:2782-2790). There are no viabletreatment regimens for androgen-independent prostate cancer.

To study the progression of androgen-dependent prostate cancer toandrogen-independent prostate cancer, the human CWR22 prostate cancerxenograft model was propagated in nude mice (see Pretlow, et al. (1993)J. Natl. Cancer Inst. 85:394-398). The CWR22 xenograft isandrogen-dependent when grown in male nude mice. Androgen-independentsub-lines can be derived by first establishing androgen-dependent tumorsin male mice. The mice are then castrated to remove the primary sourceof growth stimulus (androgen), resulting in tumor regression. Within 3-4months, molecular events prompt the tumors to relapse and start growingas androgen-independent tumors. See, e.g., Nagabhushan, et al. (1996)Cancer Res. 56:3042-3046; Amler, et al. (2000) Cancer Res. 60:6134-6141;and Bubendorf, et al. (1999) J. Natl. Cancer Inst. 91:1758-1764.

We have previously monitored the gene expression changes that occurduring the transition from androgen-dependence to androgen-independenceusing the CWR22 xenograft model (see WO02098358). Tumors were grownsubcutaneously in male nude mice. Tumors were harvested at differenttimes after castration. The time points ranged from 0 to 125 dayspost-castration. Castration resulted in tumor regression. At day 120 andthereafter, the tumors relapsed and started growing in the absence ofandrogen.

Gene expression profiling of the harvested tumors was accomplished usingthe Eos Hu03 oligonucleotide microarray (Affymetrix Eos Hu03) (Henshallet al. (2003) Cancer Res. 63:4196-4203). Our results identified severalhundred genes that exhibited significant gene expression changesassociated with androgen ablation therapy. Some genes were associatedwith the androgen-dependent growth phase of the CWR22 tumors(pre-castration and 1-5 days post-castration), some genes wereassociated with the androgen-withdrawal phase (10-82 days postcastration, characterized by tumor regression and/or tumor growthstasis), and some genes were associated with the androgen-independentgrowth of CWR22 (greater than 120 days post castration). See WO02098358.From these results, we determined that the gene encoding LIV-1 is notandrogen-regulated and exhibited high expression levels inandrogen-dependent tumors and in all tumors undergoingandrogen-withdrawal experiment, including tumors that grew in anandrogen-independent manner (data not shown).

Castrated CWR22 xenograft nude male mice would be used as a model systemfor prevention of androgen-independent prostate cancer growth. CWR22tumor bearing mice would be treated, post androgen-ablation therapy(castration), with anti-LIV-1 antibody conjugated with Auristatin-E.Post-castration treatment with anti-LIV-1 conjugated with Auristatin-Eduring the androgen-withdrawal phase (10-82 days post castration) shouldresult in a delay in the onset of androgen-independent CWR22 tumorgrowth.

To accomplish this, CWR22 tumors would be grown in male immunodeficientmice for 2-3 weeks. The mice would then be castrated to induce tumorregression and entry into the androgen-withdrawal phase. Twenty dayspost-castration the tumors would be treated with anti-LIV-1 conjugatedwith Auristatin-E as described in Examples 1 and 2. A significant effectof anti-LIV-1-Auristatin-E would manifest itself in a delay in the onsetof androgen-independence (e.g., 5 months or more post castration). Thiswould suggest that androgen-ablation therapy patients with advancedstage prostate cancer would greatly benefit from treatment withhumanized anti-LIV-1 drug conjugates.

A non-significant effect of anti-LIV-1 ADC treatment would be due toseveral potential factors: (a) CWR22 xenograft tumors may be resistantto Auristatin E; (b) the tumor cells may not efficiently internalizeanti-LIV-1 ADC during the androgen-withdrawal phase; or (c) LIV-1protein expression may be significantly decreased during theandrogen-withdrawal phase. Modifications in treatment are available toaddress these issues.

As a model system for treating established androgen-independent prostatecancer, CWR22 tumor bearing mice would be treated at the time of onsetof androgen-independence with anti-LIV-1 drug conjugate. The objectivewould be to show that post-castration treatment with anti-LIV-1 drugconjugates during the emergence of androgen-independence (>120 days postcastration) would result in regression of androgen-independent CWR22tumors.

CWR22 tumors would be grown in male immunodeficient mice for 2-3 weeks.The mice would be then castrated to induce tumor regression and entryinto the androgen-withdrawal phase. Ten days after the tumors startgrowing in an androgen-independent manner, the tumors would be treatedwith anti-LIV-1 conjugated with Auristatin-E as described in Examples 1and 2. A significant effect of anti-LIV-1-Auristatin-E would manifestitself in regression of androgen-independent tumors. This would suggestthat patients that were treated with androgen-ablation therapy and thatsuffered relapse in the form of androgen-independent tumor growth andmetastasis would greatly benefit from treatment with humanizedanti-LIV-1 drug conjugate.

ATCC Deposit

Under the terms of the Budapest Treaty on the International Recognitionof the Deposit of Microorganisms for the Purpose of Patent Procedure,the hybridomas that produce antibodies BR2-14a, BR2-19a, BR2-22a, andBR2-23a (aka antibodies 14, 19, 22, and 23, respectively) were depositedwith the American Type Culture Collection (ATCC). The deposit of thehybridomas that produce antibodies BR2-14a, BR2-19a, and BR2-23a, weredeposited on Dec. 19, 2003 and accorded accession numbers PTA-5705,PTA-5706, and PTA-5707, respectively. The hybridoma that producesantibody BR2-22a was deposited on Nov. 11, 2010 and accorded accessionnumber PTA-11478. The ATCC is located at University Boulevard, Manassas,Va. 20110-2209, USA.

TABLE 1 DNA AND PROTEIN SEQUENCES OF LIV-1 (GENBANK ACCESSION NM_012319)SEQ ID NO: 1 LIV-1 DNA SEQUENCECTCGTGCCGA ATTCGGCACG AGACCGCGTG TTCGCGCCTG GTAGAGATTT CTCGAAGACACCAGTGGGCC CGTGTGGAAC CAAACCTGCG CGCGTGGCCG GGCCGTGGGA CAACGAGGCCGCGGAGACGA AGGCGCAATG GCGAGGAAGT TATCTGTAAT CTTGATCCTG ACCTTTGCCCTCTCTGTCAC AAATCCCCTT CATGAACTAA AAGCAGCTGC TTTCCCCCAG ACCACTGAGAAAATTAGTCC GAATTGGGAA TCTGGCATTA ATGTTGACTT GGCAATTTCC ACACGGCAATATCATCTACA ACAGCTTTTC TACCGCTATG GAGAAAATAA TTCTTTGTCA GTTGAAGGGTTCAGAAAATT ACTTCAAAAT ATAGGCATAG ATAAGATTAA AAGAATCCAT ATACACCATGACCACGACCA TCACTCAGAC CACGAGCATC ACTCAGACCA TGAGCGTCAC TCAGACCATGAGCATCACTC AGACCACGAG CATCACTCTG ACCATAATCA TGCTGCTTCT GGTAAAAATAAGCGAAAAGC TCTTTGCCCA GACCATGACT CAGATAGTTC AGGTAAAGAT CCTAGAAACAGCCAGGGGAA AGGAGCTCAC CGACCAGAAC ATGCCAGTGG TAGAAGGAAT GTCAAGGACAGTGTTAGTGC TAGTGAAGTG ACCTCAACTG TGTACAACAC TGTCTCTGAA GGAACTCACTTTCTAGAGAC AATAGAGACT CCAAGACCTG GAAAACTCTT CCCCAAAGAT GTAAGCAGCTCCACTCCACC CAGTGTCACA TCAAAGAGCC GGGTGAGCCG GCTGGCTGGT AGGAAAACAAATGAATCTGT GAGTGAGCCC CGAAAAGGCT TTATGTATTC CAGAAACACA AATGAAAATCCTCAGGAGTG TTTCAATGCA TCAAAGCTAC TGACATCTCA TGGCATGGGC ATCCAGGTTCCGCTGAATGC AACAGAGTTC AACTATCTCT GTCCAGCCAT CATCAACCAA ATTGATGCTAGATCTTGTCT GATTCATACA AGTGAAAAGA AGGCTGAAAT CCCTCCAAAG ACCTATTCATTACAAATAGC CTGGGTTGGT GGTTTTATAG CCATTTCCAT CATCAGTTTC CTGTCTCTGCTGGGGGTTAT CTTAGTGCCT CTCATGAATC GGGTGTTTTT CAAATTTCTC CTGAGTTTCCTTGTGGCACT GGCCGTTGGG ACTTTGAGTG GTGATGCTTT TTTACACCTT CTTCCACATTCTCATGCAAG TCACCACCAT AGTCATAGCC ATGAAGAACC AGCAATGGAA ATGAAAAGAGGACCACTTTT CAGTCATCTG TCTTCTCAAA ACATAGAAGA AAGTGCCTAT TTTGATTCCACGTGGAAGGG TCTAACAGCT CTAGGAGGCC TGTATTTCAT GTTTCTTGTT GAACATGTCCTCACATTGAT CAAACAATTT AAAGATAAGA AGAAAAAGAA TCAGAAGAAA CCTGAAAATGATGATGATGT GGAGATTAAG AAGCAGTTGT CCAAGTATGA ATCTCAACTT TCAACAAATGAGGAGAAAGT AGATACAGAT GATCGAACTG AAGGCTATTT ACGAGCAGAC TCACAAGAGCCCTCCCACTT TGATTCTCAG CAGCCTGCAG TCTTGGAAGA AGAAGAGGTC ATGATAGCTCATGCTCATCC ACAGGAAGTC TACAATGAAT ATGTACCCAG AGGGTGCAAG AATAAATGCCATTCACATTT CCACGATACA CTCGGCCAGT CAGACGATCT CATTCACCAC CATCATGACTACCATCATAT TCTCCATCAT CACCACCACC AAAACCACCA TCCTCACAGT CACAGCCAGCGCTACTCTCG GGAGGAGCTG AAAGATGCCG GCGTCGCCAC TTTGGCCTGG ATGGTGATAATGGGTGATGG CCTGCACAAT TTCAGCGATG GCCTAGCAAT TGGTGCTGCT TTTACTGAAGGCTTATCAAG TGGTTTAAGT ACTTCTGTTG CTGTGTTCTG TCATGAGTTG CCTCATGAATTAGGTGACTT TGCTGTTCTA CTAAAGGCTG GCATGACCGT TAAGCAGGCT GTCCTTTATAATGCATTGTC AGCCATGCTG GCGTATCTTG GAATGGCAAC AGGAATTTTC ATTGGTCATTATGCTGAAAA TGTTTCTATG TGGATATTTG CACTTACTGC TGGCTTATTC ATGTATGTTGCTCTGGTTGA TATGGTACCT GAAATGCTGC ACAATGATGC TAGTGACCAT GGATGTAGCCGCTGGGGGTA TTTCTTTTTA CAGAATGCTG GGATGCTTTT GGGTTTTGGA ATTATGTTACTTATTTCCAT ATTTGAACAT AAAATCGTGT TTCGTATAAA TTTCTAGTTA AGGTTTAAATGCTAGAGTAG CTTAAAAAGT TGTCATAGTT TCAGTAGGTC ATAGGGAGAT GAGTTTGTATGCTGTACTAT GCAGCGTTTA AAGTTAGTGG GTTTTGTGAT TTTTGTATTG AATATTGCTGTCTGTTACAA AGTCAGTTAA AGGTACGTTT TAATATTTAA GTTATTCTAT CTTGGAGATAAAATCTGTAT GTGCAATTCA CCGGTATTAC CAGTTTATTA TGTAAACAAG AGATTTGGCATGACATGTTC TGTATGTTTC AGGGAAAAAT GTCTTTAATG CTTTTTCAAG AACTAACACAGTTATTCCTA TACTGGATTT TAGGTCTCTG AAGAACTGCT GGTGSEQ ID NO: 2 LIV-1 PROTEIN SEQUENCEMARKLSVILILTFALSVTNPLHELKAAAFPQTTEKISPNWESGINVDLAISTRQYHLQQLFYRYGENNSLSVEGFRKLLQNIGIDKIKRIHIHHDHDHHSDHEHHSDHERHSDHEHHSDHEHHSDHNHAASGKNKRKALCPDHDSDSSGKDPRNSQGKGAHRPEHASGRRNVKDSVSASEVTSTVYNTVSEGTHFLETIETPRPGKLFPKDVSSSTPPSVTSKSRVSRLAGRKTNESVSEPRKGFMYSRNTNENPQECFNASKLLTSHGMGIQVPLNATEFNYLCPAIINQIDARSCLIHTSEKKAEIPPKTYSLQIAWVGGFTATSIISFLSLLGVILVPLMNRVFFKFLLSFLVALAVGTLSGDAFLHLLPHSHASHHHSHSHEEPAMEMKRGPLFSHLSSQNIEESAYFDSTWKGLTALGGLYFMFLVEHVLTLIKQFKDKKKKNQKKPENDDDVEIKKQLSKYESQLSTNEEKVDTDDRTEGYLRADSQEPSHFDSQQPAVLEEEEVMIAHAHPQEVYNEYVPRGCKNKCHSHFHDTLGQSDDLIHHHHDYHHILHHHHHQNHHPHSHSQRYSREELKDAGVATLAWMVIMGDGLHNFSDGLAIGAAFTEGLSSGLSTSVAVFCHELPHELGDFAVLLKAGMTVKQAVLYNALSAMLAYLGMATGIFIGHYAENVSMWIFALTAGLFMYVALVDMVPEMLHNDASDHGCSRWGYFFLQNAGMLLGFGIMLLISIFEHKIV FRINF

TABLE 2 ANTI-LIV-1 #1.7A4 DNA AND PEPTIDE SEQUENCES PROTEIN SEQUENCESSEQ ID NO: 3 Heavy chain variable domain; CDRs in bold and underlined:eiqlqqsgpelmkpgasvkisckas t y sftr y fmh wvkqshgeslewigyidpfnggtgynqkfkg katltvdkssstaymh lssltsedsavyycvt ygsdyfdy wgqgttltvssSEQ ID NO: 4 Light chain variable domain; CDRs in bold and underlined:divmtqpqkfmstsvgdrvsvtc kasqnvetdvv wyqqkpgqppkaliy sasyrhsgvpdrftgsgsgtnftltistvqsed laeyfc ggynnypft fgsgtkleiir DNA SEQUENCESSEQ ID NO: 5 Heavy chain variable domain:GAGATCCAGCTGCAGCAGTCTGGACCTGAGCTGATGAAGCCTGGGGCTTCAGTGAAGATATCTTGCAAGGCTTCTACTTACTCATTCACTAGGTACTTCATGCACTGGGTGAAGCAGAGCCATGGAGAGAGCCTTGAGTGGATTGGATATATTGATCCTTTCAATGGTGGTACTGGCTACAATCAGAAATTCAAGGGCAAGGCCACATTGACTGTAGACAAATCTTCCAGCACAGCCTACATGCATCTCAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGTAACGTATGGCTCCGACTACTTTGACTATTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA SEQ ID NO:6 LIGHT CHAIN VARIABLE DOMAIN:GACATTGTGATGACCCAGCCACAAAAATTCATGTCCACGTCTGTAGGCGACAGGGTCAGTGTCACCTGCAAGGCCAGTCAGAATGTGGAAACTGATGTAGTCTGGTATCAACAGAAACCTGGGCAACCACCTAAAGCACTGATTTACTCGGCATCCTACCGGCACAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAAATTTCACTCTCACCATCAGCACTGTACAGTCTGAAGACTTGGCAGAGTATTTCTGTCAGCAATATAACAACTATCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAATACGG

TABLE 3 Lists Of Medical Conditions LIV-1 has been found to beover-expressed in cancers of the organs listed in the Table. Therefore,targeting or inhibiting LIV-1, by any means known in the art, includingbut limited to antibodies, may be a an effective treatment for suchdiseases. bladder: carcinoma in situ, papillary carcinomas, transitionalcell carcinoma, squamous cell carcinoma breast: ductal carcinoma insitu, lobular carcinoma in situ ovary: ovarian carcinoma (e.g.,epithelial (serous tumors, mucinous tumors, endometrioid tumors), germcell (e.g., teratomas, chorio- carcinomas, polyembryomas, embryomalcarcinoma, endodermal sinus tumor, dysgerminoma, gonadoblastoma),stromal carcinomas (e.g., granulosal stromal cell tumors)), fallopiantube carcinoma, peritoneal carcinoma, leiomyoma prostate: epithelialneoplasms (e.g., adenocarcinoma, small cell tumors, transitional cellcarcinoma, carcinoma in situ, and basal cell carcinoma), carcinosarcoma,non-epithelial neoplasms (e.g., mesenchymal and lymphoma), germ celltumors, prostatic intraepithelial neoplasia (PIN), hormone independentprostate cancer, benign prostate hyperplasia, prostatitis

TABLE 4 Cell Lines For Validating Liv-1 Antibodies For Bladder AndOvarian Cancers: The following cell lines may be used to validate theeffectiveness of anti- LIV-1 antibodies in diseases involving ovariesand bladder. Experiments similar to those described in the Examplesabove, could be carried out. SW780 (bladder), OVCAR3 (ovarian), ES-2(ovarian).

It is understood that the examples described above in no way serve tolimit the true scope of this invention, but rather are presented forillustrative purposes. All publications, sequences of accession numbers,and patent applications cited in this specification are hereinincorporated by reference as if each individual publication or patentapplication were specifically and individually indicated to beincorporated by reference.

All UniGene cluster identification numbers and accession numbers hereinare for the GenBank sequence database and the sequences of the accessionnumbers are hereby expressly incorporated by reference. GenBank is knownin the art, see, e.g., Benson, D A, et al., Nucleic Acids Research26:1-7 (1998). Sequences are also available in other databases, e.g.,European Molecular Biology Laboratory (EMBL) and DNA Database of Japan(DDBJ).

TABLE 5 Liv-1 Mutant BCR4M1 cDNA and Protein SequencesBCR4 M1 cDNA (SEQ ID NO: 13)ATGGCGAGGAAGTTATCTGTAATCTTGATCCTGACCTTTGCCCTCTCTGTCACAAATCCCCTTCATGAACTAAAAGCAGCTGCTTTCCCCCAGACCACTGAGAAAATTAGTCCGAATTGGGAATCTGGCATTAATGTTGACTTGGCAATTTCCACACGGCAATATCATCTACAACAGCTTTTCTACCGCTATGGAGAAAATAATTCTTTGTCAGTTGAAGGGTTCAGAAAATTACTTCAAAATATAGGCATAGATAAGATTAAAAGAATCCATATACACCATGACCACGACCATCACTCAGACCACGAGCATCACTCAGACCATGAGCGTCACTCAGACCATGAGCATCACTCAGACCACGAGCATCACTCTGACCATGATCATCACTCTCACCATAATCATGCTGCTTCTGGTAAAAATAAGCGAAAAGCTCTTTGCCCAGACCATGACTCAGATAGTTCAGGTAAAGATCCTAGAAACAGCCAGGGGAAAGGAGCTCACCGACCAGAACATGCCAGTGGTAGAAGGAATGTCAAGGACAGTGTTAGTGCTAGTGAAGTGACCTCAACTGTGTACAACACTGTCTCTGAAGGAACTCACTTTCTAGAGACAATAGAGACTCCAAGACCTGGAAAACTCTTCCCCAAAGATGTAAGCAGCTCCACTCCACCCAGTGTCACATCAAAGAGCCGGGTGAGCCGGCTGGCTGGTAGGAAAACAAATGAATCTGTGAGTGAGCCCCGAAAAGGCTTTATGTATTCCAGAAACACAAATGAAAATCCTCAGGAGTGTTTCAATGCATCAAAGCTACTGACATCTCATGGCATGGGCATCCAGGTTCCGCTGAATGCAACAGAGTTCAACTATCTCTGTCCAGCCATCATCAACCAAATTGATGCTAGATCTTGTCTGATTCATACAAGTGAAAAGAAGGCTGAAATCCCTCCAAAGACCTATTCATTACAAATAGCCTGGGTTGGTGGTTTTATAGCCATTTCCATCATCAGTTTCCTGTCTCTGCTGGGGGTTATCTTAGTGCCTCTCATGAATCGGGTGTTTTTCAAATTTCTCCTGAGTTTCCTTGTGGCACTGGCCGTTGGGACTTTGAGTGGTGATGCTTTTTTACACCTTCTTCCACATTCTCATGCAAGTCACCACCATAGTCATAGCCATGAAGAACCAGCAATGGAAATGAAAAGAGGACCACTTTTCAGTCATCTGTCTTCTCAAAACATAGAAGAAAGTGCCTATTTTGATTCCACGTGGAAGGGTCTAACAGCTCTAGGAGGCCTGTATTTCATGTTTCTTGTTGAACATGTCCTCACATTGATCAAACAATTTAAAGATAAGAAGAAAAAGAATCAGAAGAAACCTGAAAATGATGATGATGTGGAGATTAAGAAGCAGTTGTCCAAGTATGAATCTCAACTTTCAACAAATGAGGAGAAAGTAGATACAGATGATCGAACTGAAGGCTATTTACGAGCAGACTCACAAGAGCCCTCCCACTTTGATTCTCAGCAGCCTGCAGTCTTGGAAGAAGAAGAGGTCATGATAGCTCATGCTCATCCACAGGAAGTCTACAATGAATATGTACCCAGAGGGTGCAAGAATAAATGCCATTCACATTTCCACGATACACTCGGCCAGTCAGACGATCTCATTCACCACCATCATGACTACCATCATATTCTCCATCATCACCACCACCAAAACCACCATCCTCACAGTCACAGCCAGCGCTACTCTCGGGAGGAGCTGAAAGATGCCGGCGTCGCCACTTTGGCCTGGATGGTGATAATGGGTGATGGCCTGCACAATTTCAGCGATGGCCTAGCAATTGGTGCTGCTTTTACTGAAGGCTTATCAAGTGGTTTAAGTACTTCTGTTGCTGTGTTCTGTGCTGCGTTGCCTGCTGCATTAGGTGACTTTGCTGTTCTACTAAAGGCTGGCATGACCGTTAAGCAGGCTGTCCTTTATAATGCATTGTCAGCCATGCTGGCGTATCTTGGAATGGCAACAGGAATTTTCATTGGTCATTATGCTGAAAATGTTTCTATGTGGATATTTGCACTTACTGCTGGCTTATTCATGTATGTTGCTCTGGTTGATATGGTACCTGAAATGCTGCACAATGATGCTAGTGACCATGGATGTAGCCGCTGGGGGTATTTCTTTTTACAGAATGCTGGGATGCTTTTGGGTTTTGGAATTATGTTACTTATTTCCATATTTGAACATAAAATCGTGTTTCGTATAAATTTC BCR4 M1 protein sequence (SEQ ID NO: 14)MARKLSVILILTFALSVTNPLHELKAAAFPQTTEKISPNWESGINVDLAISTRQYHLQQLFYRYGENNSLSVEGFRKLLQNIGIDKIKRIHIHHDHDHHSDHEHHSDHERHSDHEHHSDHEHHSDHDHHSHHNHAASGKNKRKALCPDHDSDSSGKDPRNSQGKGAHRPEHASGRRNVKDSVSASEVTSTVYNTVSEGTHFLETIETPRPGKLFPKDVSSSTPPSVTSKSRVSRLAGRKTNESVSEPRKGFMYSRNTNENPQECFNASKLLTSHGMGIQVPLNATEFNYLCPAIINQIDARSCLIHTSEKKAEIPPKTYSLQIAWVGGFIAISIISFLSLLGVILVPLMNRVFFKFLLSFLVALAVGTLSGDAFLHLLPHSHASHHHSHSHEEPAMEMKRGPLFSHLSSQNIEESAYFDSTWKGLTALGGLYFMFLVEHVLTLIKQFKDKKKKNQKKPENDDDVEIKKQLSKYESQLSTNEEKVDTDDRTEGYLRADSQEPSHFDSQQPAVLEEEEVMIAHAHPQEVYNEYVPRGCKNKCHSHFHDTLGQSDDLIHHHHDYHHILHHHHHQNHHPHSHSQRYSREELKDAGVATLAWMVIMGDGLHNFSDGLAIGAAFTEGLSSGLSTSVAVFCAALPAALGDFAVLLKAGMTVKQAVLYNALSAMLAYLGMATGIFIGHYAENVSMWIFALTAGLFMYVALVDMVPEMLHNDASDHGCSRWGYFFLQNAGMLLGFGIMLLISIFEHKIVFRINF

TABLE 6 Liv-1 Antibodies Number Designation 1 1.1F10 2 1.7A4 3 BR2-10b 4BR2-11a 5 BR2-13a 6 BR2-14a 7 BR2-15a 8 BR2-16a 9 BR2-17a 10 BR2-18a 11BR2-19a 12 BR2-20a 13 BR2-21a 14 BR2-22a 15 BR2-23a 16 BR2-24a 17BR2-25a

What is claimed is:
 1. A monoclonal antibody that specifically binds toa protein having the amino acid sequence of SEQ ID NO:2, wherein theantibody comprises the complementary determining regions (CDRs) of theheavy chain variable domain amino acid sequence shown in SEQ ID NO:3,and the CDRs of the light chain variable domain amino acid sequenceshown in SEQ ID NO:4; or a chimeric or humanized form thereof.
 2. Theantibody of claim 1, wherein the antibody is a humanized form of theantibody.
 3. The antibody of claim 1, which comprises a heavy chainvariable region having the amino acid sequence of SEQ ID NO:3 and alight chain variable region having the amino acid sequence of SEQ IDNO:4.
 4. The antibody of claim 1, wherein the antibody is anantigen-binding fragment.
 5. The antibody of claim 1, wherein theantibody is conjugated to an effector component.
 6. The antibody ofclaim 5, wherein the effector component is selected from the groupconsisting of a fluorescent label, a radioisotope or a cytotoxicchemical.
 7. The antibody of claim 6, wherein the cytotoxic chemical isauristatin-E or monomethyl auristatin E.
 8. A pharmaceutical compositioncomprising a pharmaceutically acceptable excipient and the antibody ofclaim
 1. 9. The pharmaceutical composition of claim 8, wherein theantibody is an antigen-binding fragment.
 10. The pharmaceuticalcomposition of claim 8, wherein the antibody is conjugated to aneffector component comprising a radioisotope or a cytotoxic chemical.11. The pharmaceutical composition of claim 10, wherein the cytotoxicchemical is auristatin-E or monomethyl auristatin E.
 12. A method oftreating an individual with prostate or breast cancer, wherein themethod comprises administering a therapeutically effective dose of theantibody of claim
 1. 13. The method of claim 12, wherein the antibody isa chimeric antibody.
 14. The method of claim 12, wherein the antibody isa humanized antibody.
 15. The method of claim 12, wherein the antibodyis an antigen-binding fragment.
 16. The method of claim 12, wherein theantibody is conjugated to an effector component comprising aradioisotope or a cytotoxic chemical.
 17. The method of claim 16,wherein the cytotoxic chemical is auristatin-E or monomethyl auristatinE.